CN104258395B - Functions and applications of nucleotide synthetase CAD in treatment of atherosclerosis - Google Patents
Functions and applications of nucleotide synthetase CAD in treatment of atherosclerosis Download PDFInfo
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Abstract
The invention discloses functions and applications of nucleotide synthetase CAD in treatment of atherosclerosis, belonging to the fields of functions and applications of genes. ApoE-/- mice and CAD-/-ApoE-/- mice are taken as experimental subjects, an atherosclerosis model is obtained through high fat diet induction, the result proves that compared with the ApoE-/- mice, the plaque area of the aorta is obviously reduced due to the CAD gene defects, the stability of sinus plaques of the aorta is enhanced, and the inflammatory response is obviously alleviated. The functions of the CAD in atherosclerosis mainly refer to the effects of promoting aorta plaque formation, particularly the effect of promoting the atherosclerosis. Aiming at the functions of the CAD, the CAD can serve as a drug target to be used for screening medicines for preventing, relieving and/or treating the atherosclerosis, and inhibitors of the CAD can be used for preparing medicines for preventing, relieving and/or treating the atherosclerosis.
Description
Technical field
The invention belongs to function and the application of gene, it is specifically related to a kind of nucleotide synthetase CAD: carbamyl phosphate synthetase II, aspartic acid ammonia transferring enzyme and dihydroora tase (carbamoyl-phosphate synthetase II, aspartate transcarbamylase, and dihydroorotase, CAD) function and application in treatment atherosclerosis, specifically CAD prevents, alleviates and/or treats application in atherosclerotic medicine in preparation.
Background technology
Cardiovascular and cerebrovascular disease be main lethal in many developed countries because of, sickness rate and fatality rate in China raise the most 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, causes a lot of cardiocerebrovasculaevents events to occur.And the coronary artery acute stenosis that causes of the rupturing 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, inflammatory and immune response locally and systemically plays an important role in atherosclerosis generation evolution, inherent immunity and adaptive immunity participate in regulating atherosclerotic lesion jointly, show as big on pathology, medium-sized artery many places speckle is formed, it is apt to occur in blood shunt, the regions such as tremulous pulse bending and arterial branch, 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.Atheromatous plaque also exists panimmunity cell, wherein most commonly seen with macrophage and T cell, in addition with a small amount of dendritic cell (Dentritic cell, 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, is mainly made up 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, and the chemistry that the mononuclear cell sticked the most locally is produced is ingratiated with the attraction of molecule and moved under inner membrance, and is divided into macrophage further.A large amount of cholesterol esters, in macrophage inner accumulated, form foam cell, and this is atherogenesis characteristic pathological physiological process in early days.Atherosclerotic is multifactor coefficient result, and 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, and blood fat reducing and anti-inflammatory treatment are current topmost remedy measures.
CAD is a kind of multifunctional enzyme, molecular weight is 243kDa, the single polypeptide of the zones of different being made up of carbamyl phosphate synthetase II (CPSase), aspartic acid ammonia transferring enzyme (ATCase) and dihydroora tase (DHOase), be the enzyme of a pyrimidine nucleotide de novo synthesis, be that nucleotide combines oligomeric structure territory 2(NOD2) interaction protein.NOD2 is expressed in macrophage, as the Figure recognition receptor of bacteria lipopolysaccharide, works the pathogenic composition deriving from antibacterial, may activate and macrophage apoptosis by regulation Nuclear factor kappa B.Due to NOD2 scalable macrophage proinflammatory activity, and macrophage atherosclerotic lesion in early days exists, therefore it is considered that CAD may play key player in the characteristic pathological physiological process of atherogenesis, but its biological function in the characteristic pathological physiological process of atherogenesis and possible mechanism of action not yet illustrate.
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.Each molecule link during utilizing genetic engineering mice to manage for Atheromatosis is studied, and to the molecular mechanism illustrated in atherosclerosis generation evolution, the molecular target finding treatment of atherosclerosis has great importance.
Summary of the invention
It is an object of the invention to determine the expression of CAD and atherosclerotic mutual relation, it is provided that one for preventing, alleviate and/or treat the new application of atherosclerotic target gene CAD.
The purpose of the present invention is achieved through the following technical solutions:
Expression and the atherosclerotic relation of the CAD that present invention determine that are as follows:
1. CAD gene knockout significantly reduces atherosclerotic plaque area
The present invention is with ApoE gene knockout (ApoE-/-) mice is with CAD/ApoE is dual-gene knocks out (CAD-/-ApoE-/-) mice is experimental subject, obtains atherosclerosis mouse model (AS) by low fat feedstuff and high lipid food diet induced respectively.Result shows ApoE-/-And CAD-/-ApoE-/-Mice passes through low fat feed diet, and aorta tree has a small amount of speckle to be formed；After high lipid food diet, plaque area dramatically increases, CAD-/-ApoE-/-The plaque area of aorta tree, aortic sinus and the brachiocephalic trunk of mice is all significantly less than ApoE-/-Mice (Fig. 1-2).
2. CAD gene knockout is obviously enhanced the stability of aortic sinus speckle
The present invention is with ApoE gene knockout (ApoE-/-) mice is with CAD/ApoE is dual-gene knocks out (CAD-/-ApoE-/-) mice is experimental subject, obtain atherosclerosis mouse model (AS) by high lipid food diet induced, the stability of aortic sinus speckle is studied.Result shows that CAD gene knockout substantially reduces content and the area of necrotic center of macrophage 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. CAD gene knockout significantly reduces inflammatory reaction
The present invention is with ApoE gene knockout (ApoE-/-) mice is with CAD/ApoE is dual-gene knocks out (CAD-/-ApoE-/-) mice is experimental subject, obtains atherosclerosis mouse model (AS) by high lipid food diet induced, the expression to the inflammatory factor of aortic sinus speckle is studied.Result shows that CAD gene knockout significantly reduces the proinflammatory factors such as ICAM-1 and IL-6 of aortic sinus speckle, increases IL-10 etc. and presses down the expression (Fig. 4) of the scorching factor.
When being understood generation atherosclerosis by result above, CAD genetic flaw decreases plaque area, strengthens the stability of aortic sinus speckle, significantly reduces inflammatory reaction.Therefore CAD has the effect promoting that atherosclerosis occurs development, prevents and treats atherosclerotic novel targets for research and New Policy provides theoretical foundation and Clinical Basis.
Therefore, CAD gene as drug target, can build In vitro cell model or the animal model of CAD gene overexpression, is used for screening prevention, alleviate and/or treating atherosclerotic medicine；CAD gene also can design and prepare prevention as the target gene in gene therapy, alleviate and/or treat atherosclerotic medicine and/or biological reagent, is reached prevention by technique for gene engineering, is alleviated and/or treat atherosclerotic purpose.Such as with CAD as target gene, design may interfere with double-strand siRNA that CAD expresses, and after being chemically synthesized, is injected into human body and makes CAD gene silencing treat atherosclerosis by the method that RNA disturbs；Can also design and build the mutant of CAD, enter cell, the substrate specificity of competition CAD original shape after injection, thus suppress the function of CAD, play therapeutic purposes；In addition, In vitro cell model or the animal model of CAD gene overexpression with CAD for shot design micromolecular compound inhibitor, can also be utilized, by screening, find wherein can the molecule of specificity suppression CAD, thus provide new therapeutic molecules for atherosclerotic treatment.
Above-mentioned functions for CAD, it is provided that CAD prevents as drug targets in screening, alleviates and/or treat the application in atherosclerotic medicine.
Above-mentioned functions for CAD, it is provided that the inhibitor of CAD prevents in preparation, alleviates and/or treat the application in atherosclerotic medicine.
One is prevented, is alleviated and/or treat atherosclerotic medicine, comprises the inhibitor of CAD.
The inhibitor of described CAD is preferably the rna interference vector of siRNA, CAD gene of CAD gene, the antibody of CAD and other can suppress the one in the inhibitor that CAD expresses.
The present invention has such advantages as relative to prior art and effect:
1. present invention discover that the New function of CAD gene, i.e. CAD can promote atherosclerotic effect.
2. promote atherosclerotic function based on CAD, provide target for developing atherosclerotic medicine.
3. the inhibitor of CAD can be used for preparation prevention, alleviates and/or treat atherosclerotic medicine.
Accompanying drawing explanation
Fig. 1 is ApoE-/-And CAD-/-ApoE-/-The aorta tree oil red O stain of mice and plaque area cartogram, result shows that CAD gene knockout significantly reduces the plaque area (NS: there was no significant difference) of aorta tree.
Fig. 2 is ApoE-/-And CAD-/-ApoE-/-Mouse aorta hole and brachiocephalic trunk HE dyeing and plaque area add up block diagram, and result shows that CAD gene knockout significantly reduces plaque area (*: the p ＜ 0.05 vs HFD ApoE of aortic sinus-/-Group).
Fig. 3 is ApoE-/-And CAD-/-ApoE-/-Necrotic center, collagen component, macrophage and the dyeing of smooth muscle cell content and result statistics block diagram in the aortic sinus speckle of mice, result shows that CAD gene knockout substantially reduces content and the area of necrotic center of macrophage in aortic sinus speckle, increases content and collagen component content (*: the p ＜ 0.05 vs HFD ApoE of smooth muscle cell in speckle-/-Group).
Fig. 4 is ApoE-/-And CAD-/-ApoE-/-The expression immunofluorescence dyeing of the inflammatory factor such as ICAM-1, IL-6, IL-10 and result statistics block diagram in the aortic sinus speckle of mice, result shows that CAD gene knockout significantly reduces the expression of proinflammatory factor ICAM-1 and IL-6 of aortic sinus speckle, increases expression (*: the p ＜ 0.05 vs HFD ApoE of anti-inflammatory factors IL-10-/-Group).
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 to this.
Animal for research and raising
Laboratory animal kind, sex, week old and source: ApoE gene knockout (ApoE-/-) mice is with CAD/ApoE is dual-gene knocks out (CAD-/-ApoE-/-) mice, male, 8 week old, body weight 19-25g.ApoE knock out mice (ApoE-/-, purchased from Jackson
Laboratory, article No. 002052)；CAD-/-ApoE-/-Mice is obtained by CAD knock out mice (purchased from RIKEN company of Japan, article No.: RBRC01723) and the hybridization of ApoE knock out mice.
Laboratory animal feed formula: high lipid food (HFD, purchased from Fukang bio tech ltd of Beijing China, 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 Beijing China, article No. D12450B): percent of calories: protein 20%, carbohydrate 70%, fat 10%.
Animal feeding and environmental condition: all of experiment mice is 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 packet: select 8 week old, body weight 19-25g, male, ApoE-/-Mice and CAD
-/-ApoE-/-Mice, gives high lipid food (Western respectively
Diets, HFD) and low fat feedstuff (Normal chow, NC) raising, ApoE-/-HFD group, ApoE-/- NC group, CAD -/-ApoE-/-
HFD group, CAD -/-ApoE-/-
NC group totally 4 groups, often organize each 20.
2. Atherosclerosis Model is by high lipid food induction operating process:
Use ApoE-/-Mice and CAD-/-ApoE-/-Mice, sets up AS model, carries out phenotype correlation analysis, specifies the effect that CAD gene pairs atheromatosis plays.Mice is from the beginning of 8 week old, and the omnidistance high lipid food of HFD group is fed 28 weeks and put to death and collect sample, 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 end tissue sampling eventually
Mice feeds high fat or low fat feedstuff until when 28 weeks, weighing, using 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 right auricle, the syringe needle of transfusion device is lunged left ventricle, slowly inject 10-15mL PBS 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.Mice is placed under microscope, separate fascia, fatty tissue around aortic arch, cut brachiocephalic trunk, put into equipped with in the 5mL EP pipe of 4% paraformaldehyde, heart is cut in ascending aorta initial part, cut off in the middle part of thoracic aorta, and cut off at about 3mm under neck summation clavicle, 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) dyeing 60min → 60% isopropyl alcohol 1min × 3 time are to removing remaining outer wall fat → be laid on black dissection stencil plate by tremulous pulse dye under 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 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 spilling from embedding frame gap.Flowing water puts into dewaterer after rinsing 30min, 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 that Y stands in paraffin, and 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
nullHaematoxylin eosin stains (HE dyeing): take 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% 1 minute → pure water of ethanol rinsing (not hanging with the globule on slide as standard) → get rid of the moisture on most microscope slide，Haematoxylin solution (Zhuhai shellfish rope，BA-4021) 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 is contaminated，Magnesium sulfate 2g，Distilled water 100mL) 1 minute → tap water embathes (removing the Scott liquid on slide) → get rid of the moisture being all on slide，Yihong solution (Zhuhai shellfish rope，BA-4024) 10 seconds → pure water of dyeing rinsing (removing the loose colour in Yihong on slide) → 70% ethanol once → 95% ethanol once → 100% ethanol 30 seconds，3 times → dimethylbenzene 2 minutes，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.
5. brachiocephalic trunk plaque area measures
Haematoxylin eosin stains (HE dyeing), takes brachiocephalic trunk paraffin white tiles, and 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 can substantially assess that atheromatous plaque on whole piece blood vessel formed number, distribution situation and the size of plaque area.Fig. 1 is mice AS model aorta posterior tree oil red O stain result figure, and brachiocephalic trunk and aortic sinus are the most obvious positions of atheromatous plaque, ApoE-/-And CAD-/-ApoE-/-Mice passes through low fat feed diet, and aorta tree just has a small amount of speckle to be formed, and CAD-/-ApoE-/-The plaque area of mice and ApoE-/-Mice is without significant difference；After high fat diet, plaque burden dramatically increases, CAD-/-ApoE-/-The aorta tree plaque area of 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 CAD after high fat diet-/-ApoE-/-The aortic sinus of mice and the plaque area of brachiocephalic trunk are significantly less than ApoE-/-Mice.Result above shows that CAD gene knockout significantly reduces atherosclerotic plaque area.
Embodiment 3
The mensuration of AS model mice plaque stability
1. the size of aortic sinus necrotic center measures
Haematoxylin eosin stains (HE dyeing), method, with embodiment 2.4, is chosen the tissue microscope containing cholesterol crystal, acellular core fibre structure and 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, mainly comprise the following steps: take aortic sinus paraffin white tiles 55 DEG C 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 and drips in tissue, dye in wet box 90min → 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 the most dry coverslip mounting immediately of dimethylbenzene, microscope is taken pictures.
Collagen ratio measuring: use Image-Pro
Plus 6.0 image analysis software circle redness area of collagen, collagen ratio (%)=area of collagen/plaque area * 100%.
3. the expression of aortic sinus macrophage and smooth muscle cell mark measures
Immunofluorescence dyeing detection aortic sinus macrophage marker CD68, smooth muscle cell mark SMA(Smooth Muscle Actin) expression.Required anti-information a: 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: aortic sinus paraffin white tiles 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 a certain amount of pH6.0 citrate antigen retrieval working solution in repairing in box, put in pressure cooker, big fire is heated to boiling, tissue slice after dewaxing hydration is placed in reparation 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) ddH2O rinses 5min × 2 time, and PBS rinses 5min × 2 time.
6) groupization stroke circle, drips 10% sheep blood serum (GTX27481, GeneTex) and closes, and closes 60min for 37 DEG C in wet box.
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, and 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
Positive Cell Counts is measured by Plus 6.0 image analysis software, CD68/SMA(%)=positive cell number/plaque area * 100%.
Macrophage is most important cell component in speckle, differentiate after it is subcutaneous in mainly having blood circulation mononuclear cell to enter, Monocytes/Macrophages can secrete multiple adhesion, chemotactic factor such as cell adhesion molecule (ICAM-1), MCAF (MCP-1) etc., promote the entrance 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 is to repair 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, has the impact of anti-blood flow and prevents the effect of plaque rupture, is also the important evaluation index safeguarding plaque stability.
Fig. 3 is ApoE-/-Mice and CAD-/-ApoE-/-The analysis result figure of the aortic sinus speckle inclusions of mice.The visible CAD of aortic sinus HE dyeing-/-ApoE-/-The necrotic center area of mice is significantly less than ApoE-/-Mice；PSR coloration result shows the model after high fat diet, CAD-/-ApoE-/-The collagen ratio of group mice is apparently higher than ApoE-/-Mice；Immunofluorescence sends out observation macrophage marker CD68 at CAD-/-ApoE-/-Expression in mice speckle is then substantially less than ApoE-/-Mice；Smooth muscle cell mark SMA is at CAD-/-ApoE-/-Expression in mice speckle is apparently higher than ApoE-/-Mice.Result above shows that CAD gene knockout substantially reduces content and the area of necrotic center of macrophage in aortic sinus speckle, increases content and the collagen component content of smooth muscle cell in speckle；CAD 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
The expression of the inflammatory factors such as immunofluorescence dyeing detection aortic sinus 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 3.3.
Fluorescent quantitation is added up: uses Image-Pro Plus 6.0 image analysis software that positive cell carries out absorbance (IOD) and measures.
Inflammatory reaction is one of principal element of causing atheromatous plaque to rupture.The a large amount of cytokine of proinflammatory cytokine secretion such as interleukin-6 (IL-6), interleukin 10 (IL-10) etc. may result in the activation of endotheliocyte, the hypertrophy apoptosis of smooth muscle cell and the progress of medicated porridge sample pathological changes.Adhesion, chemotactic factor that impaired endotheliocyte and Monocytes/Macrophages are secreted are the key factors that transmitting inflammation cell is assembled to atheromatous plaque.The expression being observed the inflammatory factors such as ICAM-1, IL-6, IL-10 by immunofluorescence dyeing is changed, result shows that CAD gene knockout significantly reduces the expression of proinflammatory factor ICAM-1, IL-6 in aortic sinus speckle, increases IL-10 and presses down the expression (Fig. 4) of inflammatory factor.
Above-described embodiment result shows, ApoE-/-Mice and CAD-/-ApoE-/-Mice issues lively pulse atherosclerosis, and ApoE in the induction of high fat diet-/-Mice is compared, and the dual-gene Aortic Plaque area knocking out rear mice of CAD/ApoE substantially reduces, and plaque stability also increases, and inflammatory reaction substantially alleviates.These results indicate that CAD can remarkably promote the formation of Aortic Plaque and atherosclerotic develop.Present invention demonstrates that CAD has important deterioration effect in Atherosclerosis Model, its inhibitor can be used for the medicine of preparation treatment atheromatosis.
Above-described embodiment is the present invention preferably embodiment; but embodiments of the present invention are also not restricted to the described embodiments; 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 (3)
1. nucleotide synthetase CAD gene prevents as drug targets in screening, alleviates and/or treat the application in atherosclerotic medicine.
2. the inhibitor of nucleotide synthetase CAD prevents in preparation, alleviates and/or treat the application in atherosclerotic medicine.
Application the most according to claim 2, it is characterised in that: the inhibitor of described CAD is rna interference vector or the antibody of CAD of siRNA, CAD gene of CAD gene.
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| The Nucleotide Synthesis Enzyme CAD Inhibits NOD2 Antibacterial Function in Human Intestinal Epithelial Cells;Amy L. Richmond et al;《Gastroenterology》;20120630;第142卷(第7期);1483–1492 * |
| 模式识别受体在动脉粥样硬化中的作用及相互关系;王莉莉等;《中国动脉硬化杂志》;20121231;第20卷(第10期);951-955页 * |
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