CN103558392A - Anti-atherosclerosis drug efficacy evaluation method based on inflammatory responses - Google Patents
Anti-atherosclerosis drug efficacy evaluation method based on inflammatory responses Download PDFInfo
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Abstract
The invention discloses an anti-atherosclerosis drug efficacy evaluation method based on inflammatory responses, wherein the method comprises the following steps: step 1, planting smooth muscle cells on the outer side of a Millicell@insert PE (Poly Ethylene) membrane, and turning after the cells are adhered to the wall for 8 hours; step 2, planting endothelial cells on the inner side of the membrane, and culturing for 6 days totally; step 3, inoculating mononuclear cells THP-1, adding oxLDL, IL-1 and texted drugs, and culturing for 24 hours; step 4, detecting any one of the items of detecting contents of MCP-1 and TNFa in the endothelial cells; detecting the content of CD36 on the surface of the mononuclear cells; detecting the contents of MDA and MMP2 in the smooth muscle cells, observing the change of a smooth muscle cytoskeleton; observing the expression of ICAM-1 of the endothelial cells.
Description
Technical field:
The present invention relates to a kind of method of evaluating drug effect of medicine, particularly a kind of antiatherosclerotic effect evaluation method based on inflammatory reaction.
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 are thus theoretical, have explained comparatively all sidedly the formation evolution of AS.Think at present, AS is exactly a chronic inflammatory process from developing into the overall process lapsing to, numerous inflammatory cells and inflammatory mediator are participated, but inflammation participates in the mechanism of action of AS process to be illustrated so far not yet completely, and research at present mainly concentrates on effect and the mutual relationship aspect of various inflammatory cells, inflammatory mediator.
In AS focus, there is the main cells such as endothelial cell, macrophage, lymphocyte, smooth muscle cell and mast cell, study at present more and with the comparatively close inflammatory cell of AS generation and development relation be monocyte/macrophage and lymphocyte.
In AS pathogenic process, the participation of complement system has also promoted inflammatory reaction.Now in AS focus, found C1～9 complement proper constituent such as grade and multiple complement activation fragment, especially the whole last membrane attack complex C5b9 of complement activation (MAC) is present in all endarterium and fibrous plaque that thicken.CD59 a kind ofly the Complement Regulatory Protein in surface of cell membrane with glycosyl-phosphatidyl inositol (GPI) anchor, and it is bringing into play key effect in regulating MAC dress group.Research recently shows, compared with control group, compare CD59 and the process acceleration of knock-out mice generation AS altogether of ApoE gene, and this effect is weakened when endothelial cell overexpression CD59, researcher confirms that MAC has short AS effect, CD59 suppresses AS, and it may be a kind of anti-AS therapy that prompting inhibition MAC forms.
Along with going deep into of research, found that increasing inflammatory mediator has participated in generation and the development of AS.Various inflammatory cells are interrelated by inflammatory mediators such as relevant cell factor, adhesion molecules, interaction, thereby formed complicated network, and inflammatory reaction has been amplified in their cascades, has jointly promoted the progress of AS.As: c reactive protein (CRP), adhesion molecule, MCP-1, IL-6, TNF-α, salusins, except above-mentioned inflammatory mediator, have been found that at present with the closer inflammatory mediator of AS relation and also have IL-2, IL-4, IL-10, IL-18, IFN-γ, TGF-β etc.
AS is from initial morbidity to occurring that the whole process of clinical cardiovascular events, inflammatory reaction all plays an important role, yet people also know little about it for the concrete Function and its mechanisms that numerous link rose of this complex network at present.In addition,, although in some AS animal models, all obtain certain curative effect for the anti-inflammatory treatment of a plurality of links of inflammation, still lack clinically the large-scale experimental study of system.
Inflammatory reaction forms 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, developed a kind of medicaments sifting model, to antiatherogenic medicine being carried out to medicine efficacy screening by a kind of simple and practical method, the method of cultured cell in vitro for the method, can carry out on a large scale medicine primary dcreening operation, to new drug development, provide a kind of new effective screening technique.
The present invention is directed to complicated iuntercellular mutual relationship in AS formation and inflammatory reaction process, the Millicell@insert of take is carrier, the method that adopts EC-SMC-MC to cultivate altogether, observation is the interaction of three kinds of cells and the change of the characteristic of function thereof under AS risk factor oxLDL and IL-1 β stimulation, with this, build new method and the appraisement system of AS medicine drug efficacy study, for intervene the drug research of AS from inflammation angle, provide effective tool.
Summary of the invention:
The present invention is a kind of antiatherosclerotic effect evaluation method based on inflammatory reaction, 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 need to be passed through following steps:
Step 1, by smooth muscle cell, to plant the PE film outside in Millicell@insert, 8h overturns after cell attachment;
Step 2, at the medial surface plantation endothelial cell of film, cultivates 6 days altogether;
Step 3, inoculation monocyte THP-1, adds oxLDL, and IL-1 and tested medicine, cultivate 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; MDA in smooth muscle cell, the content of MMP2, observes the variation 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 to the PE film outside in Millicell@insert, after 8h, overturn.
Described step 2, method is as follows:
After upset, at the inner side of the PE film of Millicell@insert plantation endothelial cell, cultivate altogether 6 days.
Described step 3, method is as follows:
With 1 * 10
5/ cm
2inoculation monocyte THP-1, adds oxLDL, and IL-1 β and tested medicine, cultivate 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 Millicell@insert, 3 times repeatedly, then exhaust supernatant, centrifugal, get supernatant, detect EC layer MCP-1, TNF α;
The content method that detects onthe surface of monocytes CD36 is as follows:
What after centrifugal, obtain is precipitated as monokaryon THP-1 cell precipitation, is resuspended in 1ml PBS, and flow cytometer detects 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; Millicell@insert membrane immunofluorescence dyeing method of drawing material is as follows:
Take out Millicell@insert, PBS cleans insert internal membrane, outer side form each 3 times, with knife blade, film is cut gently along insert edge, and every group of 2 insert internal membrane upward, upward, bonding die agent is fixed on film on microslide 2 outer side forms of insert.
The expression of observing endothelial cell ICAM-1 is as follows:
Insert internal membrane is that EC confluent monolayer cells dropping PBS50 μ l washes 1 time, exhausts after PBS, drips PE-ICAM-1 antibody 10 μ l, and in incubator, black out is hatched after 30min, and PBS cleans 2 times, and laser confocal microscope 575nm detects ICAM-1 expression.
The changing method of observing smooth muscle cell skeleton is as follows:
The outer side form of Insert is layer of smooth muscle cells with the fixing 10min of 3.7% paraformaldehyde, acetone dehydration 1min, under 0.2%TritonX100 room temperature, permeate 10min, use again twice of PBS fine laundering, drip 10 μ g/ml FITC-phalloidine 20 μ l, in incubator, black out is hatched 30min, and PBS cleans 2 times, the variation 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, can adopt the method for bibliographical information 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 wherein explained as follows of relevant title term:
Vascular smooth muscle cell: vascular smooth muscle (vascular smooth muscle) refers to the particular type smooth muscle that is present in vascular wall and forms its major part.The cell that forms smooth muscle is called vascular smooth muscle cell.
HASMC: mankind's arteria umbilicalis smooth muscle cell (Human Aortic Smooth Muscle Cells).
Plantation: the process by cell suspension inoculation on culture medium surface.
The PE film outside of Millicell@insert: Millicell@insert is a brand of cultivating altogether cell, and PE film is the title material of the film of common cultivation cell bottom inoculating cell.Cultivating altogether cell is a unsettled little culturing room standing in culture plate, and the film medial and lateral of culturing room's bottom can inoculating cell, and a side is down called outside.
The PE film inner side of Millicell@insert: cultivate altogether a cell side upward, be called inner side.Cell attachment: cell from suspended state to whole stretching, extensions, be attached to the process culture medium.Endothelial cell: endothelial cell is the special epithelial cell of skim, is comprised 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 arteria umbilicalis endothelial cell (Human Umbilical Artery Endothelial Cells).
Cell is cultivated: it is a kind of technology that cell is cultivated (Cell culture), is eucaryote or prokaryote are cultivated under in check state, makes its growth.The development of this technology and method, in close relations with tissue cultivation or organ culture.Nutrient solution adopts DMEM, RPMI-1640 (Gibicol) nutrient culture media, and the amount that adds nutrient culture media is the culture plate 0.6ml/ of 0.4ml/ Kong， lower floor hole, Millicell insert upper strata.
Monocyte: monocyte (Monocyte) is a kind of leucocyte in human immune system.It has two kinds of effects in human immune system: supplement macrophage and the dendritic cell under normal condition 1.; 2. having under inflammation signal, monocyte can gather infected tissue fast at 8 to 12 hours, and differentiates 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 (Oxidized low-density lipoprotein).
IL-1: interleukin 1 (Interleukin-1).
Tested medicine: observed object in experiment.
Detect the MCP-1 in endothelial cell, the content of TNF α: MCP-1 (monocyte chemoattractant protein-1), monocyte chemoattractant protein, can chemotactic blood monocytes raising to inflammation part.TNF α, TNF, is a kind ofly can directly kill tumour cell, can bring into play by activating immune system the cell factor of antitumor action again.With MCP-1 in kit detection cell and TNF α, can evaluate the effect of medicine to cellular inflammation reaction.
Detect the content of onthe surface of monocytes CD36: CD36, category-B scavenger receptor, mainly expresses at onthe surface of monocytes.The expression that detects onthe surface of monocytes CD36 can be evaluated the ability of monocytic cell ingests lipid.
Detect the MMP2 in smooth muscle cell, the content of MDA: matrix metalloproteinase (matrix metalloproteinase, 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, with the crosslinked toxic effect of lipoprotein, affects key enzyme activity in mitochondrial respiratory chain cpd and mitochondria in vitro.Detect MMP2, the variation of MDA can be evaluated the impact of smooth muscle cell on plaques stabilize.
Observe the variation of smooth muscle cell skeleton: cytoskeleton concept is the nuclear skeleton-nuclear lamina system existing in nucleus.Nuclear skeleton, nuclear lamina and median fiber structurally interconnect, through nucleus and cytoplasmic rack system.The variation of observing smooth muscle cell skeleton can be evaluated the degree of cell cellular contraction under pathological state.
Observe the expression of endothelial cell ICAM-1: ICAM-1(ICAM-1), being called again CD54, belonging to the member in immunoglobulin superfamily in adhesion molecule (IGSF), is an important adhesion molecule of mediation Adhesion.ICAM-1 is low expression level on the vascular endothelial cell (VEC) of tranquillization, by the specific receptor on Surface of Vascular Endothelial Cells, is combined and is brought into play its biologic activity.
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: adopt alcohol extract, extract to prepare by the method for enriching and purifying macroporous resin, contain tanshinone IIA 3%, tanshin polyphenolic acid B 38%, andrographolide 20% in extract.
By method of the present invention, ginseng lotus extract is tested, its result is as follows:
With experimental verification in body, AS is formed and inflammatory reaction to have the ginseng lotus extract of sure curative effect be example, the Millicell@insert EC-SMC-MC co-culture system of take is instrument, from cell, molecular level further investigated, joins lotus extract to the effect of AS inflammatory reaction and mechanism.Confirm that ginseng lotus extract can THP-1 membrane molecule CD36 expresses, adhesive function by affecting; Affect HUVEC membrane molecule ICAM-1 expression, TNF α, MCP-1 secretion, HUSMC cytoskeleton is expressed; A plurality of links such as MMP2, MMP9 secretion that affect HUSMC are intervened AS processes and inflammatory reaction, corresponding with experimental result in early stage body.
The checking of ginseng lotus extract drug effect
1, the impact that ginseng lotus extract is expressed THP-1 membrane molecule CD36 in co-culture system
Adopt Flow cytometry.Cell adds 37 ℃ of pretreatments of medicine of variable concentrations and imposes IL-1 β (final concentration 10ng/ml) or ox-LDL(final concentration 100 μ g/ml) irritation cell, optimized the PE-CD3610 μ l tiring rear adding respectively, room temperature lucifuge is hatched 30min, PBS washes 3 times, re-suspended cell, the impact that Flow cytometry ginseng lotus extract is expressed film CD36.
Result shows, with control group comparison, model group THP-1 surface C D36 the positive expression rate obviously increases, and SL group obviously suppresses CD36 and expresses (P<0.01).
The impact that table 1 ginseng lotus extract is expressed THP-1 membrane molecule CD36 in co-culture system
2, the impact that ginseng lotus extract sticks THP-1 in co-culture system
Adopt calcein-AM fluorescent marker method.In every 4ml2.0 * 10
6in THP-1 cell suspension, add calcein-AM37 ℃ of 40 μ l hatch 1 hour standby.Add the 37 ℃ of pretreatment EC-SMC1h of medicine that first use variable concentrations before 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 with the comparison of blank group, has significant difference (P<0.01).Ginseng lotus extract group adherent cell number obviously declines, and relatively has significant difference (P<0.01 or P<0.05) with model group.
Fig. 1 join lotus extract EC-SMC-MC is cultivated altogether in THP-1 adhere to affect THP-1 adherent cell number, calcein-AM fluorescence labeling, laser confocal microscope detects.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 groups.
3, the impact of ginseng lotus extract on HUVEC membrane molecule expression in co-culture system and EC secretion inflammatory factor
Adopt laser co-focusing and ELISA method to detect.EC layer exhausts supernatant (approximately 400 μ l) to 4 ℃ of centrifugal 10min of 2500rpm in 1ml EP pipe, gets supernatant, detects EC layer NO, TNF α.EC confluent monolayer cells (the about 0.15cm of area
2) drip PBS50 μ l and wash 1 time, exhaust after PBS, drip PE-ICAM-1 antibody 10 μ l, in incubator, black out is hatched after 30min, and 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 in culture supernatant altogether, MCP-1, content obviously increase.Ginseng lotus extract group can reduce EC surface ICAM-1 to be expressed, and is total to TNF α in culture supernatant, and MCP-1 content, relatively has significant difference (P<0.05 or P<0.01) with model group.
A confocal detection result, enlargement factor * 100.A, b, c, d expresses (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 groups) for cultivating altogether PE mark EC surface ICAM-1 after 24h.
Table 2 is joined lotus extract to TNF α in HUAEC, the impact that MCP-1 expresses
4, the impact of ginseng lotus extract on HUSMC membrane molecule expression in co-culture system and SMC secretion inflammatory factor
Adopt laser co-focusing and ELISA method to detect.Draw nutrient culture media (approximately 600 μ l) in SMC layer culture plate, 4 ℃ of 2500rpm centrifuging and taking supernatants in 1ml EP pipe, for detection of SMC layer MMP2, MMP9.
SMC confluent monolayer cells (the about 0.15cm2 of area) drips PBS50 μ l and washes 1 time, exhausts after PBS, drips FITC-F-actin antibody 10 μ l, and in incubator, black out is hatched after 30min, and PBS cleans 2 times, and laser confocal microscope 488nm detects F-actin and expresses.
Result shows, obviously enhancing of EC-SMC-MC co-culture model group SMC F-actin expression, and MMP2 in common culture supernatant, the content of MMP9 obviously increases.Ginseng lotus extract group can reduce SMC surface F-actin to be expressed, and is total to MMP2 in culture supernatant, and MMP9 content, relatively has significant difference (P<0.05 or P<0.01) with model group.
Fig. 3 EC-SMC and EC-SMC-MC cultivate altogether and A confocal detection result, enlargement factor * 100 are compared in the effect of the different stimulated factor.E, f, g, h expresses (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 groups) for cultivating altogether FITC mark SMC cell F-actin after 24h
The impact that table 3 ginseng lotus extract is expressed MMP-9 in HUSMC
By method of the present invention, the medicine of unknown effects to be detected is detected, according to measurement result, can judge whether unknown effects medicine has study of anti-atherogenic effect.In testing process, use blank and positive drug contrast, can draw easily the drug effect result of medicine to be measured.
The maximum feature of this model is in an experimental system, to complete the evaluating drug effect of antiatherosclerosis and inflammatory reaction thereof.
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, and the system of setting up the evaluation of an antiatherosclerotic effect from inflammation angle is the core of this patent.
Accompanying drawing explanation:
Fig. 1 join lotus extract EC-SMC-MC is cultivated altogether in PE mark EC surface ICAM-1 express
Fig. 2 join lotus extract EC-SMC-MC is cultivated altogether in the impact expressed of SMC cell F-actin
Fig. 3 EC-SMC and EC-SMC-MC cultivate and the effect comparison of stimulating factor oxLDL+IL-1 altogether
Embodiment:
Further illustrate by the following examples the present invention.
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 Millicell@insert, 8h overturns after cell attachment, medial surface at film is planted EC(HUAEC with 1 * 105/cm2 kind), cultivate altogether 6d, with 1 * 105/cm2 inoculation monocyte THP-1, add oxLDL100 μ g/ml, IL-1 β 10ng/ml and be subject to reagent to cultivate 24h simultaneously.In this system, carry out pharmacodynamic experiment.
After 1.224h, with rifle head, gently draw nutrient culture media in Millicell@insert, 3 times repeatedly, then exhaust supernatant (approximately 400 μ l) to 4 ℃ of centrifugal 10min of 2500rpm in 1ml EP pipe.Get supernatant, frozen.For detection of EC layer MCP-1, TNF α.
1.3 are precipitated as monokaryon THP-1 cell precipitation, are resuspended in 1ml PBS, and flow cytometer detects onthe surface of monocytes CD36.
1.4 draw nutrient culture media (approximately 600 μ l) in lower floor's culture plate, 4 ℃ of centrifugal 10min of 2500rpm in 1ml EP pipe.Get supernatant, frozen.For detection of SMC layer MMP2, MDA.
1.5 take out Millicell@insert, PBS cleans insert internal membrane, outer side form each 3 times, with knife blade, film is cut gently along insert edge, and every group of 2 insert internal membrane upward, upward, bonding die agent is fixed on film on microslide 2 outer side forms of insert.
1.6EC confluent monolayer cells (the about 0.15cm2 of area) drips PBS50 μ l and washes 1 time, exhausts after PBS, drips PE-ICAM-1 antibody 10 μ l, and in incubator, black out is hatched after 30min, and PBS cleans 2 times, and laser confocal microscope 575nm detects ICAM-1 and expresses.
1.7SMC confluent monolayer cells (the about 0.15cm2 of area) 3.7% paraformaldehyde is 10min fixedly, and acetone dehydration 1min, permeates 10min under 0.2%TritonX100 room temperature, then use twice of PBS fine laundering.10 μ g/ml FITC-phalloidine 20 μ l, in incubator, black out is hatched 30min, and PBS cleans 2 times, the variation of laser confocal microscope 488nm observation of cell skeleton.
2 set up model and existing model comparative experiments
Cultivate and to exhaust supernatant (approximately 400 μ l) after 24h to 4 ℃ of centrifugal 10min of 2500rpm in 1ml EP pipe.Get supernatant, detect EC layer MCP-1, TNF α, the results are shown in Table 4.Draw nutrient culture media (approximately 600 μ l) in lower floor's culture plate, 4 ℃ of centrifugal 10min of 2500rpm in 1ml EP pipe.Get supernatant, detect SMC layer MMP2, MDA, the results are shown in Table 5.Get film, the 10 μ g/ml FITC-phalloidine dyeing of SMC confluent monolayer cells, observation of cell skeleton, the results are shown in Figure 3.
The different condition of culture altogether of table 4 are to EC emiocytosis MCP-1, the impact of TNF α
The different condition of culture altogether of table 5 are to SMC emiocytosis MMP2, the impact of MDA
Embodiment 2
1 unknown drug study
Tanshinone IIA (Tan IIA)
With EC-SMC-MC cultivation group altogether in contrast, with the positive contrast of Atorvastatin, according to embodiment 1 method, detect, testing result is as follows:
1.1 tanshinone IIAs are to EC emiocytosis MCP-1, and the result that affects of TNF α shows, EC-SMC-MC cultivates altogether and impose oxLDL and IL-1 stimulates, EC surface ICAM-1, and TNF α in culture supernatant altogether, and the content of MCP-1 obviously increases.Tanshinone IIA can reduce EC surface ICAM-1 expresses, and is total to TNF α in culture supernatant, MCP-1 content, and, model group relatively has significant difference (P<0.05 or P<0.01).
Table 5 tanshinone IIA is to EC emiocytosis MCP-1, the impact of TNF α
The result that affects that 1.2 tanshinone IIAs are expressed onthe surface of monocytes CD36 shows, EC-SMC-MC cultivates altogether and impose oxLDL and IL-1 stimulates, and MC surface C D36 expresses and obviously increases.Tanshinone IIA can reduce MC surface C D36 expresses, and model group relatively has 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
1.3 tanshinone IIAs are to SMC emiocytosis MMP2, and the result that affects of MDA shows, EC-SMC-MC cultivates altogether and impose oxLDL and IL-1 stimulates, and SMC F-actin expresses and obviously strengthens, MDA in culture supernatant altogether, and the content of MMP2 obviously increases.Tanshinone IIA can reduce SMC cell F-actin expresses, and is total to TNF α in culture supernatant, MCP-1 content, and model group relatively has significant difference (P<0.05 or P<0.01).
Table 7 tanshinone IIA is to SMC emiocytosis MMP2, the impact of MDA
Claims (10)
1. the antiatherosclerotic based on inflammatory reaction is imitated evaluation method, it is characterized in that described method comprises, adopt oxLDL and IL-1 as stimulating factor, act on endothelial cell-smooth muscle cell-monocyte (EC-SMC-MC) co-culture system, with the evaluating drug effect model that described method is set up, there is obvious atherosclerotic inflammatory reaction feature.
2. a method for building up for the effect of the antiatherosclerotic based on inflammatory reaction evaluation model, is characterized in that, process following steps:
Step 1, by smooth muscle cell, to plant the PE film outside in Millicell@insert, 8h overturns after cell attachment;
Step 2, at the medial surface plantation endothelial cell of film, cultivates 6 days altogether;
Step 3, inoculation monocyte THP-1, adds oxLDL, and IL-1 and tested medicine, cultivate 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; MDA in smooth muscle cell, the content of MMP2, observes the variation of smooth muscle cell skeleton; Observe the expression of endothelial cell ICAM-1.
3. according to the method for claim 2, it is characterized in that, described step 3, method is as follows:
With 1 * 10
5/ cm
2inoculation monocyte THP-1, adds oxLDL, and IL-1 β and tested medicine, cultivate 24h.
4. according to the method for claim 2, it 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 Millicell@insert, 3 times repeatedly, then exhaust supernatant, centrifugal, get supernatant, detect EC layer MCP-1, TNF α.
5. according to the method for claim 2, it is characterized in that, described step 4, wherein,
The content method that detects onthe surface of monocytes CD36 is as follows:
What after centrifugal, obtain is precipitated as monokaryon THP-1 cell precipitation, is resuspended in 1ml PBS, and flow cytometer detects onthe surface of monocytes CD36.
6. according to the method for claim 2, it 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. according to the method for claim 2, it is characterized in that, described step 4, wherein,
Millicell@insert membrane immunofluorescence dyeing method of drawing material is as follows:
Take out Millicell@insert, PBS cleans insert internal membrane, outer side form each 3 times, with knife blade, film is cut gently along insert edge, and every group of 2 insert internal membrane upward, upward, bonding die agent is fixed on film on microslide 2 outer side forms of insert.
8. according to the method for claim 2, it is characterized in that, described step 4, wherein,
The expression of observing endothelial cell ICAM-1 is as follows:
Insert internal membrane is that EC confluent monolayer cells dropping PBS50 μ l washes 1 time, exhausts after PBS, drips PE-ICAM-1 antibody 10 μ l, and in incubator, black out is hatched after 30min, and PBS cleans 2 times, and laser confocal microscope 575nm detects ICAM-1 expression.
9. according to the method for claim 2, it is characterized in that, described step 4, wherein,
The changing method of observing smooth muscle cell skeleton is as follows:
The outer side form of Insert is layer of smooth muscle cells with the fixing 10min of 3.7% paraformaldehyde, acetone dehydration 1min, under 0.2%TritonX100 room temperature, permeate 10min, use again twice of PBS fine laundering, drip 10 μ g/ml FITC-phalloidine 20 μ l, in incubator, black out is hatched 30min, and PBS cleans 2 times, the variation 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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| CN107629997A (en) * | 2017-10-13 | 2018-01-26 | 中国科学院大学 | External foam wanshing model and its application in atherosclerosis study |
| CN108546750A (en) * | 2018-04-12 | 2018-09-18 | 邦世（苏州）生物医药科技有限公司 | A kind of research method that TLR9 is applied to treatment coronary atherosclerosis |
| CN115369070A (en) * | 2022-09-05 | 2022-11-22 | 西南交通大学 | In vitro atherosclerotic plaque model, preparation method and application |
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