CN104324390B - The application in treatment atherosclerosis of the Mindin gene - Google Patents

Abstract

The present invention discloses a kind of Mindin gene function in atherosclerosis and application, belongs to function and the application of gene.The present invention is with ApoE^‑/‑Mice and Mindin^‑/‑ApoE^‑/‑Mice is experimental subject, obtains Atherosclerosis Model by high fat diet induction, and result shows and ApoE^‑/‑Mice contrasts, and Mindin genetic flaw significantly reduces the plaque area of aorta, strengthens the stability of aortic sinus speckle, significantly reduces inflammatory reaction.This shows Mindin function in atherosclerosis, is mainly reflected in Mindin and has the effect promoting that Aortic Plaque is formed, and particularly Mindin can promote atherosclerotic effect.For the above-mentioned functions of Mindin, Mindin can be used for screening prevention, alleviate and/or treating atherosclerotic medicine as drug targets, and the inhibitor of Mindin can be used for preparation prevention, alleviates and/or treat atherosclerotic medicine.

Description

The application in treatment atherosclerosis of the Mindin gene

Technical field

The invention belongs to function and the application of gene, be specifically related to a kind of Mindin in treatment atherosclerosis Function and application, specifically Mindin preparation prevention, alleviate and/or treat in atherosclerotic medicine apply.

Background technology

Cardiovascular and cerebrovascular disease be main lethal in many developed countries because of, in the sickness rate of China and fatality rate also Raise year by year.The basis of cardiovascular and cerebrovascular disease is atherosclerosis (Atheosclersisis, AS), and atherosclerosis can Make ductus arteriosus wall thicken, hardening, luminal stenosis, cause a lot of cardiocerebrovasculaevents events to occur.And atherosclerosis unstable plaque Coronary artery acute stenosis and obturation that the rupturing of block, hematoblastic gathering and thrombosis cause are to cause acute coronary The major reason of syndrome (acute coronary syndrome, ACS).

Atherosclerosis is a kind of chronic inflammation that multiple gene, risk factor and immunologic mechanism participate in jointly Disease, inflammatory and immune response locally and systemically plays an important role in atherosclerosis generation evolution, intrinsic exempts from Epidemic disease and adaptive immunity participate in regulating atherosclerotic lesion jointly, and pathology shows as big, medium-sized artery many places speckle formation, Being apt to occur in the regions such as blood shunt, tremulous pulse bending and arterial branch, its characteristics of lesion is that in blood, lipid deposits at endarterium, draws Playing inner membrance stove fibrous thickening, focus deep is the medicated porridge sample material formed by slough and extracellular lipid pond.Tremulous pulse medicated porridge Sample plaque also exists panimmunity cell, wherein most commonly seen with macrophage and T cell, in addition with a small amount of tree Prominent shape cell (Dentritic cell, DC), natural killer cell (natural killer cell, NK cell) and hypertrophy are thin Born of the same parents (mast cell) etc., occasionally have bone-marrow-derived lymphocyte.

The macroscopic damage of atherosclerosis earliest period is fatty streaks, mainly by intake of the huge of a large amount of cholesterol Phagocyte source foam cell is constituted.Mononuclear cell in blood circulation is attached to reactive endothelial cells at tremulous pulse damageable zone, Starting the formation of fatty streaks, the chemistry that the mononuclear cell sticked the most locally is produced is ingratiated with the attraction of molecule and is moved to interior Under film, and it is divided into macrophage further.A large amount of cholesterol esters, in macrophage inner accumulated, form foam cell, and this is Pulse atherosclerosis forms characteristic pathological physiological process in early days.Atherosclerotic is multifactor coefficient knot Really, the atherosclerosis risk sexual factor having now been found that is a lot, but related pins is the most undesirable to treatment and control effect, fall Fat and anti-inflammatory treatment are current topmost remedy measures.

Mindin is also called Spondin 2, belongs to Mindin-F-Spondin extracellular matrix protein family, in 1997 Cloned in Brachydanio rerio first by Higashijima etc., and find that it can promote the adhesion of hippocampal neuron and growth.Subsequently Research confirms that Mindin wide expression comprises an aminoterminal F base plate react egg in each tissue of mammal, its structure The platelet factor4 type repetitive sequence domain of (F-spondin, FS) domain and a carboxyl terminal in vain (thrombospondin type 1 repeat, TSR).Mindin between different genera structure height guard, such as mice and The homology of the Mindin aminoacid sequence of people reaches 85%, and prompting Mindin is likely to be of important biological function.Mindin is A kind of pattern recognition molecule, it is possible to be combined with pathogen and PAMPs, and as the phagocytosis merit of a kind of phagocytosis element regulation and control macrophage Can, participate in regulating autarcetic host defense.Research finds that Mindin is combined with neutrophilic granulocyte by integrin, adjusts Control the raising of neutrophilic granulocyte, adhere to and shift function；And dendritic cell that Mindin gene knockout makes derived from bone marrow is medium and small Molecule G-protein Rac-1 and the down-regulated expression of Rac-2, cause its activating T cell ability impaired, and Mindin remain by whole Close element to be combined with dendritic cell and the regulation and control of signal transduction in mediated cell；Analyze the protein structure of Mindin further, really The structure having determined Mindin identification PAMPs is TSR domain, and F-spondin domain is then the knot of Mindin and integrin Close site.Increasing research proves that Mindin not only plays a significant role in immunomodulating and inflammatory reaction, but also Participate in the pathological process of multiple disease.We there are some researches show Mindin at myocardial hypertrophy, apoplexy and diabetes conditions mould Playing an important role in type: in myocardial hypertrophy model, Mindin is as the endogenous protection factor, by suppression AKT/ GSK3 signal beta path, thus the heart failure that suppression causes because maladjustment causes cardiac remodeling (Bian ZY etl, Disruption of mindin exacerbates cardiac hypertrophy and fibrosis. J Mol Med (Berl) (2012)；90 (8): 895-910)；In ischemic brain injury, Mindin by Akt signal path thus plays biology Learn function (Wang L etl, Mindin is a critical mediator of ischemic brain injury in An experimental stroke model.Exp Neurol(2013)；247:506-16)；In diabetes model, Mindin by with PPAR α effect, thus regulate the Liver Lipid Metabolism of mice and alleviate fatty degeneration of liver, obesity, inflammation Deng (Zhu LH et al, Mindin/Spondin 2 inhibits hepatic steatosis, insulin resistance, and obesity via interaction with peroxisome proliferator- Activated receptor α in mice. J Hepatol(2014)；60 (5): 1046-54)；Based on the studies above base Plinth, it is believed that Mindin may play important angle in the characteristic pathological physiological process of atherogenesis early stage Color, but its biological function in atherogenesis characteristic pathological physiological process in early days and possible effect machine System not yet illustrates.

Mice and genetic engineering mice resource are the fields such as current disease mechanisms research, gene functional research, medicine initiative One of most important animal model, is also the necessary condition of these field innovation researches.Utilize genetic engineering mice for tremulous pulse Each molecule link during atherosis pathology is studied, to illustrating dividing in atherosclerosis generation evolution Handset system, the molecular target finding treatment of atherosclerosis has great importance.

Summary of the invention

For solving defect and the deficiency of above-mentioned prior art, the primary and foremost purpose of the present invention is to determine the table of Mindin gene Reach and atherosclerotic mutual relation, it is provided that one is used for preventing, alleviate and/or treat atherosclerotic target gene The new application of Mindin.

The purpose of the present invention is achieved through the following technical solutions:

Expression and the atherosclerotic relation of the Mindin gene that present invention determine that are as follows:

1. Mindin gene knockout significantly reduces atherosclerotic plaque area

The present invention with ApoE gene knockout (ApoE-/-) mice dual-gene with Mindin/ApoE knock out (Mindin-/- ApoE-/-) mice is experimental subject, obtains atherosclerosis mouse model (AS) by high lipid food diet induced.Result Show ApoE-/-and Mindin-/-ApoE-/-mice by after high lipid food diet, Mindin-/-ApoE-/-mice Aorta tree and aortic sinus plaque area are all significantly less than ApoE-/-mice, and body weight is without significant difference (Fig. 1-3).

2. Mindin gene knockout is obviously enhanced the stability of aortic sinus speckle

The present invention with ApoE gene knockout (ApoE-/-) mice dual-gene with Mindin/ApoE knock out (Mindin-/- ApoE-/-) mice is experimental subject, obtains atherosclerosis mouse model (AS) by high lipid food diet induced, to master The stability of tremulous pulse hole speckle is studied.Result show Mindin gene knockout substantially reduce aortic sinus speckle in huge bite thin The content of born of the same parents, increases content and the collagen component content of smooth muscle cell in speckle, enhances the stability of aortic sinus speckle (Fig. 4-5).

3. Mindin gene knockout significantly reduces inflammatory reaction

The present invention with ApoE gene knockout (ApoE-/-) mice dual-gene with Mindin/ApoE knock out (Mindin-/- ApoE-/-) mice is experimental subject, obtains atherosclerosis mouse model (AS) by high lipid food diet induced, to master The expression of the inflammatory factor of tremulous pulse hole speckle is studied.Result shows that Mindin gene knockout significantly reduces aortic sinus speckle The proinflammatory factors such as ICAM-1 and IL-6 of block, increase IL-10 etc. and press down the expression (Fig. 6) of the scorching factor.

When being understood generation atherosclerosis by result above, Mindin genetic flaw decreases plaque area, strengthens main The stability of tremulous pulse hole speckle, significantly reduces inflammatory reaction.Therefore Mindin have promotion atherosclerosis develop Effect, prevent and treat atherosclerotic novel targets and New Policy for research and provide theoretical foundation and Clinical Basis.

Therefore, Mindin gene as drug target, can build the In vitro cell model of Mindin gene overexpression or dynamic Object model, is used for screening prevention, alleviate and/or treating atherosclerotic medicine；Mindin gene also can be controlled as gene Target gene in treatment, designs and prepares prevention, alleviate and/or treat atherosclerotic medicine and/or biological reagent, logical Cross technique for gene engineering to reach prevention, alleviate and/or treat atherosclerotic purpose.Such as with Mindin as target gene, Design may interfere with double-strand siRNA that Mindin expresses, and after being chemically synthesized, is injected into what human body was disturbed by RNA Method makes Mindin gene silencing to treat atherosclerosis；Can also design and build the mutant of Mindin, after injection Enter cell, the substrate specificity of competition Mindin original shape, thus suppress the function of Mindin, play therapeutic purposes；Additionally, also may be used With with Mindin for shot design micromolecular compound inhibitor, utilize the In vitro cell model of Mindin gene overexpression or dynamic Object model, by screening, find wherein can the molecule of specificity suppression Mindin, thus carry for atherosclerotic treatment For new therapeutic molecules.

Above-mentioned functions for Mindin, it is provided that Mindin prevents as drug targets in screening, alleviates and/or treat Application in atherosclerotic medicine.

Above-mentioned functions for Mindin, it is provided that the inhibitor of Mindin prevents in preparation, alleviates and/or treat tremulous pulse The atherosis application in medicine.

One is prevented, is alleviated and/or treat atherosclerotic medicine, comprises the inhibitor of Mindin.

The inhibitor of described Mindin is preferably the rna interference vector of siRNA, Mindin gene of Mindin gene, The antibody of Mindin and other can suppress the one in the inhibitor that Mindin expresses.

The present invention has such advantages as relative to prior art and effect:

1. present invention discover that the New function of Mindin gene, i.e. Mindin can promote atherosclerotic effect.

2. promote atherosclerotic function based on Mindin, provide target for developing atherosclerotic medicine Mark.

3. the inhibitor of Mindin can be used for preparation prevention, alleviates and/or treat atherosclerotic medicine.

Accompanying drawing explanation

Fig. 1 be ApoE-/-and the aorta tree oil red O stain of Mindin-/-ApoE-/-mice and plaque area add up Figure, result shows that Mindin gene knockout significantly reduces the plaque area of aorta tree.

Fig. 2 be ApoE-/-and Mindin-/-ApoE-/-mice HFD diet after the body weight cartogram of 28 weeks, result shows 2 The body weight zero difference of group mice.

Fig. 3 is ApoE-/-and Mindin-/-ApoE-/-mouse aorta hole HE dyeing and plaque area statistics block diagram, Result show Mindin gene knockout significantly reduce aortic sinus plaque area (*: p ＜ 0.05vs HFD ApoE-/- Group).

Fig. 4 be ApoE-/-and Mindin-/-ApoE-/-mice aortic sinus speckle in collagen component content colored graph, Result shows that Mindin gene knockout significantly increases collagen contents (* *: the p ＜ 0.01vs HFD of aortic sinus ApoE-/-group).

Fig. 5 is ApoE-/-and the aorta macrophage of Mindin-/-ApoE-/-mice and smooth muscle cell content dye Chromatic graph, result shows that Mindin gene knockout substantially reduces the content of macrophage in aortic sinus speckle, flat in increasing speckle Content (* *: the p ＜ 0.01vs HFD ApoE-/-group of sliding myocyte；In figure, m represents aortic tunica media, and A represents speckle, L generation Table aorta tube chamber).

Fig. 6 be ApoE-/-and Mindin-/-ApoE-/-mice aortic sinus speckle in ICAM-1, IL-6, IL-10 Etc. expression immunofluorescence dyeing figure and the fluorescent quantitation statistics block diagram of inflammatory factor, result shows Mindin gene knockout Significantly reduce the expression of proinflammatory factor ICAM-1 and IL-6 of aortic sinus speckle, increase anti-inflammatory factors IL-10 Expression (*: p ＜ 0.05vs HFD ApoE-/-group；In figure, m represents aortic tunica media, and A represents speckle, and L represents actively Vessel lumen).

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 do not limit In this.

Animal for research and raising

Laboratory animal kind, sex, week old and source: ApoE gene knockout (ApoE-/-) mice is double with Mindin/ApoE Gene knockout (Mindin-/-ApoE-/-) mice, male, 8 week old, body weight 19-25g.ApoE knock out mice (ApoE-/-, purchased from Jackson Laboratory, article No. 002052)；Mindin-/-ApoE-/-mice is by Mnk1 clpp gene Except mice is (by Duke University Medical center immunology (Department of Immunology, Duke University Medical Center) how there is literary composition structure, and friendship is given.) and ApoE knock out mice hybridization obtain (above-mentioned Mindin base Because knock-out mice manufactures with reference to documents below: You-Wen He et al, The extracellular matrix protein mindin is a pattern-recognition molecule for microbial pathogens. Nature Immunology (2004), 5 (1): 88-97).

Laboratory animal feed formula: (HFD, purchased from Fukang bio tech ltd of Beijing China, by AIN-76 for high lipid food A Western Diets formula, percent of calories: protein 15.8%, fat 40%, carbohydrate 44.2%).

Animal feeding and environmental condition: all of experiment mice is all raised in 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 packet: select 8 week old, body weight 19-25g, male, ApoE-/-mice and Mindin-/- ApoE-/-mice, gives high lipid food (Western Diets, HFD) respectively and raises, ApoE-/-HFD group, and Mindin-/- ApoE-/-HFD group totally 2 groups, often organize each 20.

2. Atherosclerosis Model is by high lipid food induction operating process:

Use ApoE-/-mice and Mindin-/-ApoE-/-mice, set up AS model, carry out phenotype correlation analysis, bright The effect that really Mindin gene pairs atheromatosis plays.Mice is from the beginning of 8 week old, and the omnidistance high lipid food of HFD group is fed Support 28 weeks and put to death and collect sample.

Embodiment 2 AS model mice plaque area measures

1. mice end tissue sampling eventually

Mice feeds high fat or low fat feedstuff until when 28 weeks, weighing, and uses 3% pentobarbital sodium, 90mg/kg anesthetized mice, It is fixed on material drawing board with syringe needle, moistens mice skin of chest abdomen with gauze, cut off thoracic cavity with eye scissors, expose heart, cut off the right side Auricle, lunges left ventricle the syringe needle of transfusion device, slowly injects 10-15mL PBS with 50mL syringe, treats right auricle Effluent is limpid, changes 4% paraformaldehyde and continues to inject 10-15mL.After perfusion terminates, remove splanchnocoel internal organs, only retain the heart Dirty.Mice is placed under microscope, separates fascia, fatty tissue around aortic arch, cut brachiocephalic trunk, put into equipped with 4% poly In the 5mL EP pipe of formaldehyde, cut heart in ascending aorta initial part, cut off in the middle part of thoracic aorta, and under neck summation clavicle Cut off at about 3mm, aortic arch is put in above-mentioned EP pipe.

2. aorta tree plaque area measures

Aorta tree is placed in fixing → pure water rinsing 30min → 60% isopropanol overnight in 4% paraformaldehyde and processes 10 min → oil red O dye liquor (company sigma, article No. 00625) dyes 60 min → 60% isopropyl alcohol 1min × 3 time to clean background Remove remaining outer wall fat → tremulous pulse dye is laid in black under → anatomical lens and dissect on stencil plate, after dyeing, use number phase Machine is taken pictures, and uses Image-Pro Plus 6.0 image analysis software to carry out plaque area quantitative determination (oil red O stock solution=0.5 Gram oil red O+100 milliliter 100% isopropanol, 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 processes

Prepared by 3.1 paraffin specimens

In 4% paraformaldehyde overnight fixing after, brachiocephalic trunk, aortic arch filter paper are carefully wrapped, in case between embedding frame Gap spills.Flowing water puts into dewaterer after rinsing 30min, and 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 brachiocephalic trunk and aortic arch have been dehydrated, take out from dewaterer.Brachiocephalic trunk is Y Standing in paraffin, aortic arch lies low in paraffin.

3.2 aortic tissue sections

Microtome is used respectively aortic sinus and brachiocephalic trunk paraffin specimen to be cut into slices (slice thickness 5 μm).

4. aortic sinus plaque area measures

Haematoxylin eosin stains (HE dyeing): take aortic sinus paraffin white tiles 65 DEG C 30 minutes → dimethylbenzene of baking 5 minutes × 3 times → 100% ethanol 1 minute → 95% ethanol 1 minute → 70% 1 minute → pure water of ethanol rinses (not hang with the globule on slide For standard) → getting rid of the moisture on most microscope slide, haematoxylin solution (Zhuhai shellfish rope, BA-4021) contaminates 5 minutes → tap water rinse (removing haematoxylin loose colour on slide) → 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 (to be removed on slide Scott liquid) → get rid of the moisture being all on slide, Yihong solution (Zhuhai shellfish rope, BA-4024) dye 10 seconds → pure water rinsing (removing the loose colour in Yihong on slide) → 70% ethanol once → 95% ethanol once → 100% ethanol 30 seconds, 3 times → dimethylbenzene 2 points Clock, 3 times → take advantage of dimethylbenzene the most dry time mounting (with bubble-free of cutting into slices as principle) → fume hood in dry up, microscope is taken pictures.

HE dyeing picture statistics: directly with Image-Pro Plus 6.0 image analysis software circle aortic sinus speckle face Long-pending.

By aorta tree oil red O stain can substantially assess that atheromatous plaque on whole piece blood vessel formed number, Distribution situation and the size of plaque area.Fig. 1 is oil red O stain result figure after mice AS model, and brachiocephalic trunk and aortic sinus are The most obvious position of atheromatous plaque, ApoE-/-pass through high lipid food diet with Mindin-/-ApoE-/-mice After, the aorta tree plaque area of Mindin-/-ApoE-/-mice is significantly less than ApoE-/-mice, and body weight is without significant difference (Fig. 2).Fig. 3 is mice AS model posterior sinus of Valsalva HE coloration result figure, result show after high fat diet Mindin-/- The plaque area of the aortic sinus of ApoE-/-mice is significantly less than ApoE-/-mice.Result above shows Mindin gene knockout Significantly reduce atherosclerotic plaque area.

The mensuration of embodiment 3 AS model mice plaque stability

1. aortic sinus collagen component assay:

Picro-Sirius red (PSR) dyes, and mainly comprises the following steps: 55 DEG C of baking 30min → dimethylbenzene 2min, 3 times → 100% ethanol 1min → 95% ethanol 1min → 70% ethanol 1min → flowing water rinses 10min → distilled water 1min → mass fraction 0.2% phosphomolybdic acid 2min → 0.1% sirius red picric acid solution drips in tissue, the 90min that dyes in wet box → remove residul liquid-removing → 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 that dimethylbenzene is the most dry to be covered immediately Slide mounting, microscope is taken pictures.

Collagen ratio (%)=area of collagen/plaque area * 100%.

2. aortic sinus macrophage and smooth muscle cell are expressed and are measured

Immunofluorescence dyeing detection macrophage marker CD68, smooth muscle cell mark SMA(Smooth Muscle Actin) expression.A required anti-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).

Mainly comprise the following steps:

1) roasting sheet: paraffin section is placed in more than 30min in baking box.

2) dewaxing: dimethylbenzene 5min × 3 time.

3) hydration: 100% ethanol 5min × 2 time；95% ethanol 5min；70% ethanol 5min；DdH2O embathes 5min × 2 time.

4) citrate tissue antigen recovery (Pressure method): take the work of a certain amount of pH6.0 citrate antigen retrieval Liquid, in repairing in box, is put in pressure cooker, and big fire is heated to boiling, is placed in reparation box by the tissue slice after dewaxing hydration, 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, from So take out section after cooling.

5) ddH₂O rinses 5min × 2 time, and PBS rinses 5min × 2 time.

6) groupization stroke circle, drips 10% sheep blood serum (GTX27481, GeneTex) and closes, 37 DEG C of closings in wet box 60min。

7) abandoning confining liquid, the one of dropping proper proportion dilution resists, 4 DEG C of overnight incubation, and 37 DEG C of rewarming 30min discard one and resist, PBS washes 10min × 3 time.

8) dropping two resists, and hatches 60min for 37 DEG C in wet box, discards two and resists, and 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, after it is subcutaneous in mainly having blood circulation mononuclear cell to enter Differentiating, Monocytes/Macrophages can secrete multiple adhesion, chemotactic factor such as cell adhesion molecule (ICAM-1), mononuclear cell Chemotactic and activity factor (MCP-1) etc., promote the entrance of speckle inner cell, and macrophage also can secrete multiple matrix metal in addition Protease (MMPs), reduces area of collagen in speckle, thus destroys 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 is to repair destroyed cellular stromal component Thus play a protective role；Collagen component is most important extracellular matrix in speckle, is also the main component of fibrous cap, has The impact of anti-blood flow prevents the effect of plaque rupture, is also the important evaluation index safeguarding plaque stability.

Fig. 4-5 is the analysis result figure of the speckle inclusions of ApoE-/-mice and Mindin-/-ApoE-/-mice.Actively The necrotic center area of the visible Mindin-/-ApoE-/-mice of arteries and veins hole HE dyeing is significantly less than ApoE-/-mice (under Fig. 3)； PSR coloration result shows the model after high fat diet, the collagen ratio of Mindin-/-ApoE-/-group mice apparently higher than ApoE-/-mice；Immunofluorescence is sent out observation macrophage marker CD68 and is expressed in Mindin-/-ApoE-/-mice speckle Amount is then substantially less than ApoE-/-mice；Smooth muscle cell mark SMA is expression in Mindin-/-ApoE-/-mice speckle Apparently higher than ApoE-/-mice.Result above shows that Mindin gene knockout substantially reduces macrophage in aortic sinus speckle Content and the area of necrotic center, increase content and the collagen component content of smooth muscle cell in speckle；Mindin clpp gene Except the stability (Fig. 4-5) that can strengthen aortic sinus speckle

The mensuration of inflammatory Cytokines Expression in embodiment 4 AS model mice speckle

The expression of the inflammatory factors such as immunofluorescence dyeing detection ICAM-1, IL-6, IL-10.Required anti-information a: 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).

Mainly comprise the following steps: see embodiment 3.2

Fluorescent quantitation is added up: use Image-Pro Plus 6.0 image analysis software that positive cell is carried out absorbance (IOD) measure.

Inflammatory reaction is one of principal element of causing atheromatous plaque to rupture.The a large amount of cell of proinflammatory cytokine secretion The factor such as interleukin-6 (IL-6), interleukin 10 (IL-10) etc. may result in the activation of endotheliocyte, smooth muscle cell Hypertrophy apoptosis and the progress of medicated porridge sample pathological changes.Impaired endotheliocyte and the adhesion of Monocytes/Macrophages secretion, chemotactic factor are The key factor that transmitting inflammation cell is assembled to atheromatous plaque.ICAM-1, IL-6 is observed by immunofluorescence dyeing , the expression change of the inflammatory factor such as IL-10, result shows that Mindin gene knockout is proinflammatory in significantly reducing aortic sinus speckle Factor ICAM-1, the expression of IL-6, increase the expression (Fig. 6) of IL-10 anti-inflammatory cytokine.

Above-described embodiment result shows, ApoE-/-mice and Mindin-/-ApoE-/-mice are in the induction of high fat diet Issue lively pulse atherosclerosis, compare with ApoE-/-mice, the dual-gene Aortic Plaque knocking out rear mice of Mindin/ApoE Area substantially reduces, and plaque stability also increases, and inflammatory reaction substantially alleviates.These results indicate that Mindin can remarkably promote The formation of Aortic Plaque and atherosclerotic develop.Present invention demonstrates that Mindin is in Atherosclerosis Model In have important deterioration effect, its inhibitor can be used for preparation prevention, alleviates and/or treat the medicine of atheromatosis Thing.

Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify, All should be the substitute mode of equivalence, within being included in protection scope of the present invention.

Claims (1)

1.Mindin gene prevents, alleviates and/or treats answering in atherosclerotic medicine as drug targets in screening With, it is characterised in that described medicine is the medicine of suppression Mindin gene expression, and described application is non-diagnostic and non-treatment 's.