CN104383561A - Function and application of signal regulatory protein 1 treating atherosclerosis - Google Patents

Abstract

The invention discloses function and application of signal regulatory protein 1 (SHPS1) treating atherosclerosis, and belongs to the field of functions and application of genes. According to the technical scheme, an ApoE<-/-> mouse and a SHPS1<-/->ApoE<-/-> mouse are taken as experiment objects, atherosclerotic models are obtained through high-fat diet induction, and results show that compared with the ApoE<-/-> mouse, SHPS1 gene defect leads to substantial reduction of the aorta plaque area, stability enhancement of aortic sinus plaque and mitigation of inflammation reaction. The results show that SHPS1 mainly has the effects of promoting formation of aorta plaque area and especially promoting atherosclerosis in the functions of atherosclerosis. Aiming at the above functions of SHPS1, SHPS1 can be used as a medicine target and is applied to screen medicines for preventing, alleviating and/or treating atherosclerosis, and an inhibitor of SHPS1 is applicable to prepare medicines for preventing, alleviating and/or treating atherosclerosis.

Description

The function and application of signal adjusting protein 1 in treatment atherosclerosis

Technical field

The invention belongs to function and the application of gene, be specifically related to a kind of signal adjusting protein 1(SHPS1) treating the function and application in atherosclerosis, specifically SHPS1 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.

Tumor necrosis factor-alpha (tumor necrosis factor-α, TNF-α) be a multifunctional cytokine, it is the main regulatory factors of apoptosis, inflammation and immunity, plays Promote cell's growth, differentiation, apoptosis and brings out the important function such as inflammation after the specific receptors bind on it and cell membrane.The imbalance of TNF-alpha signal and septicemia, diabetes, cancer, osteoporosis and atherosclerosis etc. closely related [1].Research finds, all there is the NF-κ B of activation in the various kinds of cell such as macrophage, smooth muscle cell, endotheliocyte of atherosclerotic lesion.By the mice of High-fat diet LDLR mRNA defect (LDLR-/-), the activation of nuclear Factor-Kappa B can be found in early days in the endotheliocyte of mouse aorta root, and develop into atherosclerotic plaque [2] gradually.Wang Chunbin etc. find the expression by suppressing NF-κ B in Atherosclerosis, and the generation of the inflammation-inhibiting factor can alleviate atherosclerosis reaction [3].

Signal adjusting protein (signal regulatory proteins, SIRPs) family's contactin member, first SIRP family member be found is SHPS1, namely containing Protein-tyrosine-phosphatase substrate 1(Src homology 2 (SH2) the domain-containing protein tyrosine phosphatase substrate1 of Src homeodomain 2), be also called SIRP α (signal regulatory protein α), the neural adhesion molecule [4,5] of BIT, MFR or P84.SHPS1 is the transmembrane protein of an about 115kDa, is one of SIRP family member.SHPS1 mainly expresses on myocardial cell, dendritic cell, macrophage, leukocyte, neurocyte, secondly in fibroblast, also have expression, play negativity and regulate the phagocytosis of macrophage, the activation of mastocyte, the activation of dendritic cell and promote the effect of apoptosis.Recent studies have found that SHPS1 can negativity regulate the activation of NF-κ B signal to promote the apoptosis that TNF-α induces, thus play the effect [6,7] of cell growth inhibiting and propagation.Infer thus, SHPS1 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 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、Barzilay JI, Abraham L, Heckbert SR, et al. The relation of markers of inflammation to the development of glucose disorders in the elderly: the Cardiovascular Health Study. Diabetes. 2001, 50(10): 2384-9.

2、Hajra L, Evans AI, Chen M, et al. The NF-kappa B signal transduction pathway in aorticendothelial cells is primed for activation in regions predisposed to atherosclerotic lesion formation. Proc Natl Acad Sci U S A, 2000, 97(16): 9052-9057.

3, Wang Chunbin, in tall and big, Yin Yuehui, etc.Curcumin is on the impact of hyperlipemia rabbit aorta NF-κ B, VCAM-1, vegf expression.Chinese patent medicine, 2007,29 (8): 1127-1129.

4、Kharitonenkov, Chen Z, Sures I, et al. A family of proteins that inhibit signalling through tyrosine kinase receptors. Nature.1997; 386(6621): 181-6.

5、Comu S,Weng W, Olinsky S, et al. The murine P84 neural adhesion molecule is SHPS-1, a member of the phosphatase-binding protein family. J Neurosci. 1997; 17(22): 8702-10.

6、Zhiyong Liao, Mingjiang Wu, Xiaoli Chen. Novel function of RECS1 as a negative regulator of TNF-a-induced NF-kB activation. Mol Cell Biochem. 2009.

7、Neznanov N, Neznanova L, Kondratov RV, et al. Dominant Negative Form of Signal-regulatory Protein-α(SIRPα/SHPS-1) Inhibits Tumor Necrosis Factor-mediated Apoptosis by Activation of NF-κB. The Journal of Biological Chemistry.2003; 278(6): 3809-15。

Summary of the invention

The object of the invention is to determine the expression of SHPS1 and atherosclerotic mutual relation, provide one for preventing, alleviating and/or treat the novelty teabag of atherosclerotic target gene SHPS1.

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

Expression and the atherosclerotic relation of the SHPS1 that the present invention determines are as follows:

1. SHPS1 gene knockout significantly reduces atherosclerotic plaque area

The present invention is with ApoE gene knockout (ApoE ^-/-) mice and SHPS1/ApoE is dual-gene knocks out (SHPS1 ^-/-apoE ^-/-) mice is experimental subject, obtains atherosclerosis mouse model (AS) respectively by low fat feedstuff and high lipid food diet induced.Result shows ApoE ^-/-and SHPS1 ^-/-apoE ^-/-mice is by low fat feed diet, and aorta tree has a small amount of Mottling formation; After high lipid food diet, plaque area significantly increases, SHPS1 ^-/-apoE ^-/-the aorta tree of mice, the plaque area of aortic sinus and brachiocephalic trunk are all significantly less than ApoE ^-/-mice (Fig. 1-2).

2. SHPS1 gene knockout significantly strengthens the stability of aortic sinus speckle

The present invention is with ApoE gene knockout (ApoE ^-/-) mice and SHPS1/ApoE is dual-gene knocks out (SHPS1 ^-/-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 SHPS1 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. SHPS1 gene knockout significantly reduces inflammatory reaction

The present invention is with ApoE gene knockout (ApoE ^-/-) mice and SHPS1/ApoE is dual-gene knocks out (SHPS1 ^-/-apoE ^-/-) mice is experimental subject, 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 SHPS1 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, SHPS1 genetic flaw decreases plaque area, strengthens the stability of aortic sinus speckle, significantly reduces inflammatory reaction.Therefore SHPS1 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, SHPS1 gene can be used as drug target, builds In vitro cell model or the animal model of SHPS1 gene overexpression, for screening prevention, alleviating and/or treating atherosclerotic medicine; SHPS1 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 SHPS1, design the double-strand siRNA that SHPS1 can be disturbed to express, after being synthesized by chemical method, be injected into human body and make SHPS1 gene silencing treat atherosclerosis by the method that SHPS1 disturbs; Can also design and build the mutant of SHPS1, after injection, entering cell, the substrate specificity of competition SHPS1 original shape, thus suppressing the function of SHPS1, playing therapeutic purposes; In addition, can also be shot design micromolecular compound inhibitor with SHPS1, utilize In vitro cell model or the animal model of SHPS1 gene overexpression, by screening, find wherein to suppress the molecule of SHPS1 by specificity, thus provide new therapeutic molecules for atherosclerotic treatment.

For the above-mentioned functions of SHPS1, SHPS1 is provided to prevent as drug targets in screening, alleviate and/or treat the application in atherosclerotic medicine.

For the above-mentioned functions of SHPS1, provide the inhibitor of SHPS1 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 SHPS1.

The inhibitor of described SHPS1 is preferably the rna interference vector of siRNA, SHPS1 gene of SHPS1 gene, the one in the antibody of SHPS1 and other inhibitor that SHPS1 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 SHPS1 gene, and namely SHPS1 can promote atherosclerotic effect.

2. promoting atherosclerotic function based on SHPS1, providing target for developing atherosclerotic medicine.

3. the inhibitor of SHPS1 can be used for preparation prevention, alleviates and/or treat atherosclerotic medicine.

Accompanying drawing explanation

Fig. 1 is ApoE ^-/-and SHPS1 ^-/-apoE ^-/-the aorta tree oil red O stain of mice and plaque area cartogram, result shows that SHPS1 gene knockout significantly reduces the plaque area of aorta tree.

Fig. 2 is ApoE ^-/-and SHPS1 ^-/-apoE ^-/-mouse aorta hole and brachiocephalic trunk HE dye and plaque area statistics block diagram, and result shows that SHPS1 gene knockout significantly reduces plaque area (*: the p < 0.05 vs HFD ApoE of aortic sinus and brachiocephalic trunk ^-/-group).

Fig. 3 is ApoE ^-/-and SHPS1 ^-/-apoE ^-/-the dyeing of necrotic center, collagen component, macrophage and smooth muscle cell content and result statistics block diagram in the aortic sinus speckle of mice, result shows that SHPS1 gene knockout significantly reduces the content of macrophage and the area of necrotic center 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 SHPS1 ^-/-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 SHPS1 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 pressing down scorching factor 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 thereto.

Animal for research and raising

Laboratory animal kind, sex, age in week and source: ApoE gene knockout (ApoE ^-/-) mice and SHPS1/ApoE is dual-gene knocks out (SHPS1 ^-/-apoE ^-/-) mice, male, 8 week age, body weight 19-25g.ApoE knock out mice (ApoE ^-/-, purchased from Jackson Laboratory, article No. 002052); SHPS1 ^-/-apoE ^-/-mice is obtained by SHPS1 knock out mice (purchased from Japanese RIKEN company, article No. RBRC01544) 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 SHPS1 ^-/-apoE ^-/-mice, gives high lipid food (Western Diets, HFD) respectively and low fat feedstuff (Normal chow, NC) is raised, ApoE ^-/-hFD group, ApoE ^-/-nC group, SHPS1 ^-/-apoE ^-/-hFD group, SHPS1 ^-/-apoE ^-/-nC group is totally 4 groups, often organizes each 20.

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

Adopt ApoE ^-/-mice and SHPS1 ^-/-apoE ^-/-mice, sets up AS model, carries out phenotype correlation analysis, specifies the effect that SHPS1 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(H ₂o)=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 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 SHPS1 ^-/-apoE ^-/-mice is by low fat feed diet, and aorta tree just has a small amount of Mottling formation, at this moment SHPS1 ^-/-apoE ^-/-the aorta tree plaque area of mice is significantly less than ApoE ^-/-mice; After high fat diet, plaque burden significantly increases, SHPS1 ^-/-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 SHPS1 after high fat diet ^-/-apoE ^-/-the aortic sinus of mice and the plaque area of brachiocephalic trunk are significantly less than ApoE ^-/-mice.Above result shows that SHPS1 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

Aortic sinus paraffin white tiles 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: get aortic sinus paraffin white tiles 55 DEG C 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. the expression of aortic sinus macrophage and smooth muscle cell mark measures

Immunofluorescence dyeing detects aortic sinus 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: aortic sinus paraffin white tiles 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 ApoE ^-/-mice and SHPS1 ^-/-apoE ^-/-the analysis result figure of the aortic sinus speckle inclusions of mice.Aortic sinus HE dyes visible SHPS1 ^-/-apoE ^-/-the necrotic center area of mice is significantly less than ApoE ^-/-mice; PSR coloration result shows the model after high fat diet, SHPS1 ^-/-apoE ^-/-the collagen ratio of group mice is apparently higher than ApoE ^-/-mice; Immunofluorescence is sent out and is observed macrophage marker CD68 at SHPS1 ^-/-apoE ^-/-expression in mice speckle is then remarkable in ApoE ^-/-mice; Smooth muscle cell mark SMA is at SHPS1 ^-/-apoE ^-/-expression in mice speckle is apparently higher than ApoE ^-/-mice.Above result shows that SHPS1 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; SHPS1 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 aortic sinus 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 of smooth muscle cell, apoptosis 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 SHPS1 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 SHPS1 ^-/-apoE ^-/-mice issues lively pulse atherosclerosis in the induction of high fat diet, and ApoE ^-/-mice is compared, and the dual-gene Aortic Plaque area knocking out rear mice of SHPS1/ApoE significantly reduces, and plaque stability also increases, and inflammatory reaction obviously alleviates.These results show, SHPS1 significantly can promote the formation of Aortic Plaque and atheroscleroticly to develop.Present invention demonstrates that SHPS1 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.SHPS1 prevents as drug targets in screening, alleviates and/or treat the application in atherosclerotic medicine.

The inhibitor of 2.SHPS1 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 SHPS1.

4. application according to claim 2 or medicine according to claim 3, is characterized in that: the inhibitor of described SHPS1 is the one in the rna interference vector of siRNA, SHPS1 gene of SHPS1 gene or the antibody of SHPS1 and other inhibitor that SHPS1 can be suppressed to express.