CN103558392B - A kind of effect of the antiatherosclerotic based on inflammatory reaction evaluation method - Google Patents

A kind of effect of the antiatherosclerotic based on inflammatory reaction evaluation method Download PDF

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CN103558392B
CN103558392B CN201310487504.6A CN201310487504A CN103558392B CN 103558392 B CN103558392 B CN 103558392B CN 201310487504 A CN201310487504 A CN 201310487504A CN 103558392 B CN103558392 B CN 103558392B
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朱晓新
李玉洁
杨庆
翁小刚
陈颖
王娅杰
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Abstract

Based on an antiatherosclerotic effect evaluation method for inflammatory reaction, it is characterized in that, through following steps: step 1, by smooth muscle cell to plant outside the PE film of Millicellinsert, 8h overturns after cell attachment; Step 2, at the medial surface plantation endothelial cell of film, Dual culture 6 days; Step 3, inoculation monocyte THP-1, adds oxLDL, IL-1 and test medicine, cultivates 24 hours.Step 4, detects any one in following items: detect the MCP-1 in endothelial cell, the content of TNF α; The content of onthe surface of monocytes CD36; The content of the MDA in smooth muscle cell, MMP2, observes the change of smooth muscle cell skeleton; Observe the expression of endothelial cell ICAM-1.

Description

A kind of effect of the antiatherosclerotic based on inflammatory reaction evaluation method
Technical field:
The present invention relates to a kind of method of evaluating drug effect of medicine, particularly a kind of effect of the antiatherosclerotic based on inflammatory reaction evaluation method.
Background technology:
Pathogenesis about atherosclerotic (AS) once had following several theory: lipid infiltrates theory, platelet aggregation and thrombosis theory, monoclonal smooth muscle cell proliferation theory and immunological theory and injury response theory, wherein injury response theory and the inflammation that develops thus theoretical, explain the shaping and development process of AS comparatively all sidedly.Think at present, AS is exactly a chronic inflammatory process from developing into the overall process lapsed to, numerous inflammatory cell and inflammatory mediator are participated, but the mechanism of action that inflammation participates in AS process is illustrated so far not yet completely, current research mainly concentrates on various inflammatory cell, the effect of inflammatory mediator and mutual relationship aspect.
There is the main cells such as endothelial cell, macrophage, lymphocyte, smooth muscle cell and mast cell in AS focus, at present research is more and be monocyte/macrophage and lymphocyte with the inflammatory cell that AS occurs and development relationship is comparatively close.
In AS pathogenic process, the participation of complement system also promotes inflammatory reaction.Now in AS focus, found C1 ~ 9 complement proper constituent such as grade and multiple complement activation fragment, especially complement activation eventually last membrane attack complex C5b9 (MAC) be present in all endarterium of thickening and fibrous plaque.CD59 is that one anchors the Complement Regulatory Protein in surface of cell membrane with glycosyl-phosphatidyl inositol (GPI), and it plays key effect in adjustment MAC dress group.Nearest research shows, compare CD59 and ApoE gene compared with control group and be total to the process acceleration that AS occurs knock-out mice, and this effect is weakened when endothelial cell overexpression CD59, researcher confirms that MAC has short AS effect, CD59 then suppresses AS, and it may be a kind of anti-AS therapy that prompting suppresses MAC to be formed.
Along with going deep into of research, find that increasing inflammatory mediator take part in generation and the development of AS.Various inflammatory cell is interrelated by the relevant inflammatory mediator such as cell factor, adhesion molecule, interact, thus constitute complicated network, and the inflammatory reaction of their Cascaded amplification, facilitates the progress of AS jointly.As: c reactive protein (CRP), adhesion molecule, MCP-1, IL-6, TNF-α, salusins, except above-mentioned inflammatory mediator, have been found that to also have IL-2, IL-4, IL-10, IL-18, IFN-γ, TGF-β etc. with the closer inflammatory mediator of AS relation at present.
AS is from initial onset to the whole process occurring clinical cardiovascular events, and inflammatory reaction all plays an important role, however at present people for this complex network numerous links the concrete Function and its mechanisms that rises also know little about it.In addition, although in some AS animal models, the anti-inflammatory treatment for the multiple link of inflammation all obtains certain curative effect, still lacks the large-scale experimental study of system clinically.
Inflammatory reaction is formed in atherosclerotic (AS), each stage ubiquity of development.Interaction between monocyte (MC), endothelial cell (EC), smooth muscle cell (SMC) and the aftercurrent effect of triggering are the core links of AS inflammatory reaction.
The present invention is according to the interaction relationship between above-mentioned cell, develop a kind of medicaments sifting model, to carrying out medicine efficacy screening by a kind of simple and practical method to antiatherogenic medicine, the method of the method cultured cell in vitro, medicine primary dcreening operation can be carried out on a large scale, provide a kind of effective screening technique newly to new drug development.
The present invention is directed to AS to be formed and iuntercellular mutual relationship complicated in inflammatory reaction process, with Millicellinsert for carrier, adopt the method for EC-SMC-MC Dual culture, the characteristic of the interaction and function thereof of observing three kinds of cells under AS risk factor oxLDL and IL-1 β stimulates changes, new method and the appraisement system of AS medicine drug efficacy study is built, for the drug research of intervening AS from inflammation angle provides effective tool with this.
Summary of the invention:
The present invention is a kind of antiatherosclerotic based on inflammatory reaction effect evaluation method, described method adopts oxLDL and IL-1 as stimulating factor, act in endothelial cell-smooth muscle cell-monocyte (EC-SMC-MC) co-culture system, there is obvious atherosclerotic inflammatory reaction feature.
Method of the present invention, practical operation needs through following steps:
Step 1, by smooth muscle cell to plant outside the PE film of Millicellinsert, 8h overturns after cell attachment;
Step 2, at the medial surface plantation endothelial cell of film, Dual culture 6 days;
Step 3, inoculation monocyte THP-1, adds oxLDL, IL-1 and test medicine, cultivates 24 hours.
Step 4, detects any one in following items: detect the MCP-1 in endothelial cell, the content of TNF α; The content of onthe surface of monocytes CD36; The content of the MDA in smooth muscle cell, MMP2, observes the change of smooth muscle cell skeleton; Observe the expression of endothelial cell ICAM-1.Described step 1, method is as follows:
Smooth muscle cell is planted outside the PE film of Millicellinsert, overturn after 8h.
Described step 2, method is as follows:
At the inner side of the PE film of Millicellinsert plantation endothelial cell after upset, Dual culture 6 days.
Described step 3, method is as follows:
With 1 × 10 5/ cm 2inoculation monocyte THP-1, adds oxLDL, IL-1 β and test medicine, cultivates 24h.
Described step 4, wherein,
Detect the MCP-1 in endothelial cell, the content method of TNF α is as follows:
Draw nutrient culture media in Millicellinsert, 3 times repeatedly, then exhaust supernatant, centrifugal, get supernatant, detect EC layer MCP-1, TNF α;
The content method detecting onthe surface of monocytes CD36 is as follows:
What obtain after centrifugal is precipitated as monokaryon THP-1 cell precipitation, is resuspended in 1mlPBS, flow cytomery onthe surface of monocytes CD36;
Detect the MDA in smooth muscle cell, the content method of MMP2 is as follows:
Draw nutrient culture media in lower floor's culture plate, centrifugal, get supernatant, detect MDA, MMP2 content; Millicellinsert membrane immunofluorescence dyeing method of drawing material is as follows:
Take out Millicellinsert, PBS cleans insert internal membrane, outer side form each 3 times, is cut gently by film with knife blade along insert edge, often organizes 2 insert internal membrane upward, upward, film is fixed on microslide 2 outer side forms of insert by bonding die agent.
The expression of observing endothelial cell ICAM-1 is as follows:
Insert internal membrane and EC confluent monolayer cells drip PBS50 μ l and wash 1 time, and after exhausting PBS, drip PE-ICAM-1 antibody 10 μ l, after in incubator, black out hatches 30min, PBS cleans 2 times, and laser confocal microscope 575nm detects ICAM-1 and expresses.
The changing method observing smooth muscle cell skeleton is as follows:
The outer side form of Insert and layer of smooth muscle cells 3.7% paraformaldehyde fix 10min, acetone dehydration 1min, 10min is permeated under 0.2%TritonX100 room temperature, use PBS fine laundering twice again, drip 10 μ g/mlFITC-phalloidine 20 μ l, in incubator, black out hatches 30min, and PBS cleans 2 times, the change of laser confocal microscope 488nm observation of cell skeleton;
In the present invention, method for establishing model belongs to innovative approach, and each refers to that object detection method all belongs to prior art category, and the method for bibliographical information can be adopted to detect, and in this Innovation Model, the testing result of each index can judge the validity of medicine.
The method of the invention, being explained as follows wherein about title term:
Vascular smooth muscle cell: vascular smooth muscle (vascularsmoothmuscle) refers to and is present in vascular wall and the particular type smooth muscle forming its major part.The cell forming smooth muscle is called vascular smooth muscle cell.
HASMC: mankind's TSH in omphalorrhagia (HumanAorticSmoothMuscleCells).
Plantation: by the process of cell suspension inoculation on culture medium surface.
Outside the PE film of Millicellinsert: Millicellinsert is the brand of a Dual culture cell, PE film is the title material of the film of Dual culture cell bottom inoculating cell.Dual culture cell is a unsettled little culturing room stood in culture plate, and the film medial and lateral of culturing room's bottom can inoculating cell, and side down, is called outside.
Inside the PE film of Millicellinsert: Dual culture cell side upward, be called inner side.Cell attachment: cell is from suspended state to the process all stretching, be attached to culture medium.Endothelial cell: endothelial cell is the special epithelial cell of skim, is made up of one deck pinacocyte.It forms the inwall of blood vessel, is the interface of vessel lumen inner blood and other vascular walls.
HUAEC: mankind's umbilical artery endothelium cell (HumanUmbilicalArteryEndothelialCells).
Cell chulture: cell chulture (Cellculture) is a kind of technology, is eucaryote or prokaryote are cultivated under in check state, makes it grow.The development of this technology and method, with tissue cultures or organ culture in close relations.Nutrient solution adopts DMEM, RPMI-1640 (Gibicol) nutrient culture media, and the amount adding nutrient culture media is 0.4ml/ hole, Millicellinsert upper strata, lower floor culture plate 0.6ml/ hole.
Monocyte: monocyte (Monocyte) is a kind of leucocyte in human immune system.It has two kinds of effects in human immune system: 1. supplement the macrophage under normal condition and dendritic cell; 2. having under inflammatory signals, monocyte at 8 to 12 hours rapid aggregation to infected tissue, and can differentiate macrophage and dendritic cell generation immune response.
Monocyte THP-1: the title of people's Acute Monocytic Leukemia Cell Line strain is suspension growth cell.
OxLDL: oxidized low-density lipoprotein (Oxidizedlow-densitylipoprotein).
IL-1: interleukin 1 (Interleukin-1).
Test medicine: object observed in an experiment.
Detect the MCP-1 in endothelial cell, the content of TNF α: MCP-1 (monocytechemoattractantprotein-1), monocyte chemoattractant protein, can chemotactic blood monocytes raising to inflammation part.TNF α, TNF is that one directly can kill tumour cell, plays again the cell factor of antitumor action by activating immune system.With MCP-1 and TNF α in kit detection cell, the effect that medicine reacts cellular inflammation can be evaluated.
Detect the content of onthe surface of monocytes CD36: CD36, category-B scavenger receptor, mainly expresses at onthe surface of monocytes.The expression detecting onthe surface of monocytes CD36 can evaluate the ability of monocytic cell ingests lipid.
Detect the MMP2 in smooth muscle cell, the content of MDA: matrix metalloproteinase (matrixmetalloproteinase, MMP) is a kind of Zn 2+dependent neutral protein enzyme family.Its major function is degraded and moulds extracellular matrix again, maintains the mobile equilibrium of extracellular matrix, participates in the much pathology of human body and physiology course.MDA (alondialdehyde), MDA, lipid oxidation end-product, is cross-linked toxic effect with lipoprotein, in vitro key enzyme activity in effect string mitochondrial respiratory chain compound and mitochondria.The change detecting MMP2, MDA can evaluate the impact of smooth muscle cell on plaques stabilize.
Observe the change of smooth muscle cell skeleton: cytoskeleton concept is the nuclear skeleton-nuclear lamina system existed in nucleus.Nuclear skeleton, nuclear lamina and median fiber are structurally interconnected, through nucleus and cytoplasmic rack system.The change observing smooth muscle cell skeleton can evaluate the degree of cell cellular contraction under pathological state.
Observe the expression of endothelial cell ICAM-1: ICAM-1(ICAM-1), being called CD54 again, belonging to the member in immunoglobulin superfamily in adhesion molecule (IGSF), is mediate an important adhesion molecule of Adhesion.ICAM-1 is upper in low expression level at the vascular endothelial cell (VEC) of tranquillization, is combined and plays its biologic activity by the specific receptor on Surface of Vascular Endothelial Cells.
Ginseng lotus extract: be a Chinese medicinal compound extract, its formula is as follows: the red sage root: Herba Andrographitis=15:9(W/W), its preparation method is as follows: the method preparation adopting alcohol extract, extract enriching and purifying macroporous resin, containing tanshinone IIA 3%, tanshin polyphenolic acid B 38%, andrographolide 20% in extract.
Test ginseng lotus extract by method of the present invention, its result is as follows:
The ginseng lotus extract of curative effect is certainly had to be example with experiment in vivo checking to AS formation and inflammatory reaction thereof, with MillicellinsertEC-SMC-MC co-culture system for instrument, from cell, molecular level further investigated ginseng lotus extract to the effect of AS inflammatory reaction and mechanism.THP-1 membrane molecule CD36 expresses, adhesive function by affecting to confirm ginseng lotus extract; Affect HUVEC membrane molecule ICAM-1 expression, TNF α, MCP-1 secretion, HUSMC cytoskeleton is expressed; Affect multiple links such as MMP2, MMP9 secretion of HUSMC and intervene AS process and inflammatory reaction, corresponding to experiment in vivo result in early stage.
The checking of ginseng lotus extract drug effect
1, the impact that lotus extract is expressed THP-1 membrane molecule CD36 in co-culture system is joined
Adopt Flow cytometry.Cell adds medicine 37 DEG C of pretreatments of variable concentrations and imposes IL-1 β (final concentration 10ng/ml) or ox-LDL(final concentration 100 μ g/ml) irritation cell, add the PE-CD3610 μ l having optimized and tired respectively afterwards, room temperature lucifuge hatches 30min, PBS washes 3 times, re-suspended cell, the impact that Flow cytometry ginseng lotus extract is expressed film CD36.
Result shows, compares with control group, and model group THP-1 surface C D36 the positive expression rate obviously increases, and SL group obviously suppresses CD36 to express (P<0.01).
The impact that lotus extract is expressed THP-1 membrane molecule CD36 in co-culture system joined by table 1
2, the impact that lotus extract sticks THP-1 in co-culture system is joined
Adopt calcein-AM fluorescent marker method.In every 4ml2.0 × 10 6add in THP-1 cell suspension 40 μ lcalcein-AM37 DEG C hatch 1 hour for subsequent use.Medicine 37 DEG C of pretreatment EC-SMC1h of variable concentrations are first used before adding fluorescence labeling THP-1, add THP-1 and IL-1 β (final concentration 10ng/ml) or ox-LDL(final concentration 100 μ g/ml) hatch, remove supernatant, PBS hole flushing 3 times, washes away and does not adhere to THP-1.Fluorescent microscope film making, analysis.
Result display model group adherent cell number obviously raises, and compares, have significant difference (P<0.01) with blank group.Ginseng lotus extract group adherent cell number obviously declines, and compares have significant difference (P<0.01 or P<0.05) with model group.
Fig. 1 join lotus extract in EC-SMC-MC Dual culture THP-1 adhere to affect THP-1 adherent cell number, calcein-AM fluorescence labeling, laser confocal microscope detect.A is Normal group in EC-SMC-MC co-culture system; B model group; C is Atorvastatin10ng/ml group; D is ginseng lotus extract 100 μ g/ml group.
3, join lotus extract and the impact of inflammatory factor is secreted on the expression of HUVEC membrane molecule and EC in co-culture system
Laser co-focusing and ELISA method is adopted to detect.EC layer exhausts supernatant (about 400 μ l) 4 DEG C of centrifugal 10min of 2500rpm to 1mlEP pipe, gets supernatant, detects EC layer NO, TNF α.(area is about 0.15cm to EC confluent monolayer cells 2) drip PBS50 μ l and wash 1 time, after exhausting PBS, drip PE-ICAM-1 antibody 10 μ l, after in incubator, black out hatches 30min, PBS cleans 2 times, and laser confocal microscope 575nm detects ICAM-1 and expresses.
Result shows, EC-SMC-MC co-culture model group, EC surface ICAM-1, and TNF α, MCP-1 in Dual culture supernatant, content obviously increase.Ginseng lotus extract group can reduce EC surface ICAM-1 and express, and TNF α, MCP-1 content in Dual culture supernatant, compares have significant difference (P<0.05 or P<0.01) with model group.
A confocal detection result, enlargement factor × 100.A, b, c, d are that after Dual culture 24h, PE marks EC surface ICAM-1 expression (a is control group, and b is model group, and c is Atorvastatin10ng/ml group, and d is ginseng lotus extract 100 μ g/ml group).
The impact that lotus extract is expressed TNF α, MCP-1 in HUAEC joined by table 2
4, join lotus extract and the impact of inflammatory factor is secreted on the expression of HUSMC membrane molecule and SMC in co-culture system
Laser co-focusing and ELISA method is adopted to detect.Draw nutrient culture media (about 600 μ l) in SMC layer culture plate, 4 DEG C of 2500rpm centrifuging and taking supernatants in 1mlEP pipe, for detecting SMC layer MMP2, MMP9.
SMC confluent monolayer cells (area is about 0.15cm2) drips PBS50 μ l and washes 1 time, and after exhausting PBS, drip FITC-F-actin antibody 10 μ l, after in incubator, black out hatches 30min, PBS cleans 2 times, and laser confocal microscope 488nm detects F-actin and expresses.
Result shows, EC-SMC-MC co-culture model group SMCF-actin expresses and obviously strengthens, and in Dual culture supernatant, the content of MMP2, MMP9 obviously increases.Ginseng lotus extract group can reduce SMC surface F-actin and express, and MMP2, MMP9 content in Dual culture supernatant, compares have significant difference (P<0.05 or P<0.01) with model group.
Fig. 3 EC-SMC and EC-SMC-MC Dual culture and the effect of the different stimulated factor compare A confocal detection result, enlargement factor × 100.E, f, g, h are that after Dual culture 24h, FITC marks SMC cell F-actin expression (a is control group, and b is model group, and c is Atorvastatin10ng/ml group, and d is ginseng lotus extract 100 μ g/ml group)
The impact that lotus extract is expressed MMP-9 in HUSMC joined by table 3
Detect with the medicine of method of the present invention to unknown effects to be detected, according to measurement result, can judge whether unknown effects medicine has study of anti-atherogenic effect.Use blank and positive drug control in testing process, the pharmacodynamic results of medicine to be measured can be drawn easily.
The maximum feature of this model is the evaluating drug effect that can complete antiatherosclerosis and inflammatory reaction thereof in an experimental system.
The present invention also comprises:
The application of method of the present invention in atherosclerotic evaluating drug effect
The application of method of the present invention in atherosclerosis reaction evaluating drug effect
Each refers to that object detection method is placed in an individual system, sets up an antiatherosclerotic imitate the core that the system evaluated is this patent from inflammation angle.
Accompanying drawing illustrates:
Fig. 1 joins lotus extract and marks the ICAM-1 expression of EC surface to PE in EC-SMC-MC Dual culture
Fig. 2 joins the impact that lotus extract is expressed SMC cell F-actin in EC-SMC-MC Dual culture
The effect of Fig. 3 EC-SMC and EC-SMC-MC Dual culture and stimulating factor oxLDL+IL-1 is compared
Embodiment:
Further illustrate the present invention by the following examples.
Embodiment 1
The method that 1.EC-MC-SMC co-culture system is set up:
1.1 first by SMC(HASMC) with 1 × 105/cm2(volume 50 μ l) plant outside the PE film of Millicellinsert, 8h overturns after cell attachment, EC(HUAEC is planted with 1 × 105/cm2 kind) at the medial surface of film, Dual culture 6d, monocyte THP-1 is inoculated with 1 × 105/cm2, add oxLDL100 μ g/ml simultaneously, IL-1 β 10ng/ml and by reagent cultivate 24h.Pharmacodynamic experiment is carried out in this system.
Gently draw nutrient culture media in Millicellinsert with rifle head after 1.224h, 3 times repeatedly, then exhaust supernatant (about 400 μ l) 4 DEG C of centrifugal 10min of 2500rpm to 1mlEP pipe.Get supernatant, frozen.For detecting EC layer MCP-1, TNF α.
1.3 are precipitated as monokaryon THP-1 cell precipitation, are resuspended in 1mlPBS, flow cytomery onthe surface of monocytes CD36.
1.4 draw nutrient culture media (about 600 μ l) in lower floor's culture plate, 4 DEG C of centrifugal 10min of 2500rpm in 1mlEP pipe.Get supernatant, frozen.For detecting SMC layer MMP2, MDA.
1.5 take out Millicellinsert, PBS cleans insert internal membrane, outer side form each 3 times, is cut gently by film with knife blade along insert edge, often organizes 2 insert internal membrane upward, upward, film is fixed on microslide 2 outer side forms of insert by bonding die agent.
1.6EC confluent monolayer cells (area is about 0.15cm2) drips PBS50 μ l and washes 1 time, and after exhausting PBS, drip PE-ICAM-1 antibody 10 μ l, after in incubator, black out hatches 30min, PBS cleans 2 times, and laser confocal microscope 575nm detects ICAM-1 and expresses.
1.7SMC confluent monolayer cells (area is about 0.15cm2) 3.7% paraformaldehyde fixes 10min, permeates 10min, then use PBS fine laundering twice under acetone dehydration 1min, 0.2%TritonX100 room temperature.10 μ g/mlFITC-phalloidine 20 μ l, in incubator, black out hatches 30min, and PBS cleans 2 times, the change of laser confocal microscope 488nm observation of cell skeleton.
2 Modling model and existing model comparative experiments
Supernatant (about 400 μ l) 4 DEG C of centrifugal 10min of 2500rpm to 1mlEP pipe are exhausted after cultivating 24h.Get supernatant, detect EC layer MCP-1, TNF α, the results are shown in Table 4.Draw nutrient culture media (about 600 μ l) in lower floor's culture plate, 4 DEG C of centrifugal 10min of 2500rpm in 1mlEP pipe.Get supernatant, detect SMC layer MMP2, MDA, the results are shown in Table 5.Get film, SMC confluent monolayer cells 10 μ g/mlFITC-phalloidin, observation of cell skeleton, the results are shown in Figure 3.
The different Dual culture condition of table 4 to EC emiocytosis MCP-1, the impact of TNF α
The different Dual culture condition of table 5 to SMC emiocytosis MMP2, the impact of MDA
Embodiment 2
1 unknown drug study
Tanshinone IIA (TanIIA)
With EC-SMC-MC Dual culture group in contrast, be that positive control detects according to embodiment 1 method with Atorvastatin, testing result is as follows:
The affect result of 1.1 tanshinone IIAs to EC emiocytosis MCP-1, TNF α shows, EC-SMC-MC Dual culture also imposes oxLDL and IL-1 stimulation, EC surface ICAM-1, and in Dual culture supernatant, the content of TNF α, MCP-1 obviously increases.Tanshinone IIA can reduce EC surface ICAM-1 expresses, and TNF α, MCP-1 content in Dual culture supernatant, model group compares significant difference (P<0.05 or P<0.01).
Table 5 tanshinone IIA to EC emiocytosis MCP-1, the impact of TNF α
1.2 tanshinone IIAs show the result that affects that onthe surface of monocytes CD36 expresses, and EC-SMC-MC Dual culture also imposes oxLDL and IL-1 stimulation, and MC surface C D36 expresses and obviously increases.Tanshinone IIA can reduce MC surface C D36 expresses, and model group compares significant difference (P<0.05 or P<0.01).
The impact that table 6 tanshinone IIA is expressed THP-1 membrane molecule CD36 in co-culture system
The affect result of 1.3 tanshinone IIAs to SMC emiocytosis MMP2, MDA shows, EC-SMC-MC Dual culture also imposes oxLDL and IL-1 stimulation, and SMCF-actin expresses and obviously strengthens, and in Dual culture supernatant, the content of MDA, MMP2 obviously increases.Tanshinone IIA can reduce SMC cell F-actin expresses, and TNF α, MCP-1 content in Dual culture supernatant, and model group compares significant difference (P<0.05 or P<0.01).
Table 7 tanshinone IIA to SMC emiocytosis MMP2, the impact of MDA

Claims (10)

1., based on an antiatherosclerotic effect evaluation method for inflammatory reaction, it is characterized in that described method comprises, inoculation endothelial cell, smooth muscle cell and monocyte build co-culture system, add oxLDL, IL-1 β and test medicine, cultivate 24 hours; Detect following items as being subject to reagent drug effect comprehensive evaluation index: detect the MCP-1 in endothelial cell, the content of TNF α; The content of onthe surface of monocytes CD36; The content of the MDA in smooth muscle cell, MMP2, observes the change of smooth muscle cell skeleton; Observe the expression of endothelial cell ICAM-1.
2., based on a method for building up for the antiatherosclerotic effect evaluation model of inflammatory reaction, it is characterized in that, through following steps:
Step 1, planted by smooth muscle cell outside the PE film of Millicellinsert, 8h overturns after cell attachment;
Step 2, at the medial surface plantation endothelial cell of film, Dual culture 6 days;
Step 3, inoculation monocyte THP-1, adds oxLDL, IL-1 β and test medicine, cultivates 24 hours;
Step 4, detects any one in following items: detect the MCP-1 in endothelial cell, the content of TNF α; The content of onthe surface of monocytes CD36; The content of the MDA in smooth muscle cell, MMP2, observes the change of smooth muscle cell skeleton; Observe the expression of endothelial cell ICAM-1.
3. method according to claim 2, is characterized in that, described step 3, and method is as follows:
With 1 × 10 5/ cm 2inoculation monocyte THP-1, adds oxLDL, IL-1 β and test medicine, cultivates 24h.
4. method according to claim 2, is characterized in that, described step 4, wherein,
Detect the MCP-1 in endothelial cell, the content method of TNF α is as follows:
Draw nutrient culture media in Millicellinsert, 3 times repeatedly, then exhaust supernatant, centrifugal, get supernatant, detect EC layer MCP-1, TNF α.
5. method according to claim 2, is characterized in that, described step 4, wherein,
The content method detecting onthe surface of monocytes CD36 is as follows:
Be precipitated as monokaryon THP-1 cell precipitation by what obtain after centrifugal for the upper strata nutrient culture media in Millicellinsert, be resuspended in 1mlPBS, flow cytomery onthe surface of monocytes CD36.
6. method according to claim 2, is characterized in that, described step 4, wherein,
Detect the MDA in smooth muscle cell, the content method of MMP2 is as follows:
Draw nutrient culture media in lower floor's culture plate, centrifugal, get supernatant, detect MDA, MMP2 content.
7. method according to claim 2, is characterized in that, described step 4, wherein,
Millicellinsert membrane immunofluorescence dyeing method of drawing material is as follows:
Take out Millicellinsert, PBS cleans insert internal membrane, outer side form each 3 times, is cut gently by film with knife blade along insert edge, often organizes 2 insert internal membrane upward, upward, film is fixed on microslide 2 outer side forms of insert by bonding die agent.
8. method according to claim 2, is characterized in that, described step 4, wherein,
The expression of observing endothelial cell ICAM-1 is as follows:
Insert internal membrane and EC confluent monolayer cells drip PBS50 μ l and wash 1 time, and after exhausting PBS, drip PE-ICAM-1 antibody 10 μ l, after in incubator, black out hatches 30min, PBS cleans 2 times, and laser confocal microscope 575nm detects ICAM-1 and expresses.
9. method according to claim 2, is characterized in that, described step 4, wherein,
The changing method observing smooth muscle cell skeleton is as follows:
The outer side form of Insert and layer of smooth muscle cells 3.7% paraformaldehyde fix 10min, acetone dehydration 1min, 10min is permeated under 0.2%TritonX100 room temperature, use PBS fine laundering twice again, drip 10 μ g/mlFITC-phalloidine 20 μ l, in incubator, black out hatches 30min, and PBS cleans 2 times, the change of laser confocal microscope 488nm observation of cell skeleton.
10. claim 1 or 2 method application in atherosclerotic evaluating drug effect described in any one.
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