CN108546750A - A kind of research method that TLR9 is applied to treatment coronary atherosclerosis - Google Patents
A kind of research method that TLR9 is applied to treatment coronary atherosclerosis Download PDFInfo
- Publication number
- CN108546750A CN108546750A CN201810324827.6A CN201810324827A CN108546750A CN 108546750 A CN108546750 A CN 108546750A CN 201810324827 A CN201810324827 A CN 201810324827A CN 108546750 A CN108546750 A CN 108546750A
- Authority
- CN
- China
- Prior art keywords
- cell
- coronary
- tlr9
- group
- huvec
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/323—Arteriosclerosis, Stenosis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A kind of research method that TLR9 is applied to treatment coronary atherosclerosis, the present invention relates to field of medical technology；Research object and grouping；Blood sample collection；Indexs measure：Cardiac ultrasonic；Patients with coronary heart disease is preoperative, it is postoperative, detected without myocardium enzyme of coronary disease patient, CRP, BNP；Patients with coronary heart disease preoperative and postoperative is detected without TLR9 of coronary disease patient；Statistical analysis.By the expression for detecting TLR9 mRNA in patients with coronary heart disease and healthy person peripheral blood mononuclear cells with RT PCR, specifying TLR9 and coronary heart disease and its prognosis has what kind of relationship irrelevant step analysis of going forward side by side is, the influence that Percutaneous coronary interventions express TLR9 establishes new target spot for coronary heart disease treatment.
Description
Technical field
The present invention relates to field of medical technology, and in particular to a kind of that TLR9 is applied to treatment coronary atherosclerosis
Research method.
Background technology
Atherosclerosis (Atherosclerosis, AS) is the most basic pathological change of coronary heart disease, with tube wall inner lipid
Accumulation, immunocyte (macrophage, T cell and mast cell etc.) infiltration are characterized, and early lesion is mainly shown as subendothelial
Lipid and macrophage formed foam cells composition fat line formation, as the progress of time gradually forms one by fibre
Tie up cap covering necrotic center (being made of apoptotic cell, non-viable non-apoptotic cell, cell fragment and cholesterol crystal etc.), and patch by
It is cumulative to cause luminal stenosis that cause myocardial ischemia even myocardial infarction greatly.Macrophage is as the most typical scorching in AS patches
Disease cell plays an important role during the occurrence and development of AS.Under the influence of different local microenvironments, macrophage can
Activation is different hypotype, is mainly shown as two kinds：Classical activated form (M1 types) with pro-inflammatory effect and with anti-inflammatory effect
Replacement activated form (M2 types), this is two extreme performances in a series of function hypotypes of macrophage.The polarization of macrophage
State (M1/M2) is then the outstanding feature for determining macrophage function, studies have found that coronary atherosclerosis and the external membrane of heart
Macrophage polarized state in adipose tissue is closely related.Macrophage can be by upper in the continually changing microenvironment of body
It adjusts the expression of its Toll-like receptor (toll like receptors, TLRs) and influences its activated phenotype, some researches show that TLR4
The expression of miR-155 can be induced, and miR-155 can cause the up-regulated expression of M1 type macrophages and inhibit M2 type correlation factors
The expression of ArgI.
The polarized state of macrophage (M1/M2) and its effect in AS patch occurrence and development are closely related, wherein M1
Type macrophage is primarily present in ApoE-/-In the early stage patch of mouse, and it is more common in vulnerable plaque；And M2 type macrophages
It is primarily present in advanced lesions, it is mostly related with artery plaque stability enhancing.There is the agonist CpG-ODN researches show that TLR9
Immune effector cell (macrophage and Dendritic Cells etc.) can be directly or indirectly activated to promote the Th1 classes such as IL-12 and TNF-α thin
The secretion of intracellular cytokine and induce body to establish Th1 type immune responses, and macrophage is by Th1 types relevant cell factor or lipopolysaccharides
Polarizable when activation is M1 types.At this point, Interleukin -1β (IL-1 β), nitric oxide synthase type (iNOS) and Ciita expression
Relative increase；Polarizable when macrophage is by activation such as Th2 types relevant cell factor such as IL-4 and IL-13 is M2 types, Jin Eryin
Play the up-regulated expression of the correlation factors such as FIZZ1, Arginase-1 and Ym1.At present whether macrophage can be influenced about TLR9
Activated phenotype be not fully understood.
It is most in recent years research shows that immune response and with its generation chronic inflammatory reaction in the pathogenesis of AS
Play important function.TLRs can pass through the respective components of identification pathogenic microorganism as a kind of classical pattern recognition receptors
Start the inherent immunity reaction of body, and is also played an important role in the acquired immune response.TLR9 is a kind of intracellular
Important model identification receptor, be distributed mainly on the immunocytes such as macrophage, B cell and Dendritic Cells, ligand is main
It is pathogen and itself DNA.Immunostimulatory sequence (ISS) is a kind of artificial synthesized TLR9 specific activation agent, includes mainly
Three types：CpG-ODN-A, B and c-type, ODN1826 belong to Type B CpG-ODN, can effectively activate the TLR9 of mouse monokaryon cell.
IRS is a kind of DNA sequence dna with immunoregulation effect, can effectively inhibit the activation of TLR9.Some researches show that ISS can be by swashing
TLR9 living and the balance deviation for causing Th1/Th2 type cytokines, and the activation table of Th1/Th2 type cytokines and macrophage
Type is closely related, so changing for TLR9 expressions may there are correlations with the polarized state of macrophage.
Many researchs all show that TLR4 plays an important roll in AS pathogenesis, but about TLR9 in AS pathogenesis
Effect it is also still not fully aware of.Studies have found that double antagonists of TLR7/9 can inhibit ApoE-/-Rat aorta intimal thickening and
Foam wanshing and reconstruct after weakening arterial vascular intervention；Chloroquine can change intracellular pH value and change TLR9 must
The acidic environment of palpus, plays the effect for inhibiting TLR9, and Razani etc. has found that chloroquine can play the effect of anti-AS by p53；
Lipid in the expression of all lipoprotein 3 and macrophage can be significantly increased after TLR9 agonist ODN1826 Stimulated Macrophages
Accumulation, especially triglycerides；Separately studies have found that may be promoted by NF- κ B and IRF7 approach downstream after TLR9 activation
The formation of foam cells, but it is also unclear about specific effects of the TLR9 in AS pathogenesis.
Vitro data is shown, is mediated stimulation monocyte to be divided into macrophage by CpG DNA, is made to enter under blood vessel endothelium
Low-density lipoprotein aoxidize, cause its modification to become oxidized low-density lipoprotein cholesterol, then it is thin by macrophage
Born of the same parents' scavenger receptor huge uptake causes intracellular lipid accumulation, forms Macrophage derived foamy cell.Foam cells is AS morning
The important symbol of phase, implys that TLR9 may promote the development of atherosclerosis.Sorrentino R et al. discoveries,
In vitro, including the oligodeoxynucleotide segment of CpG causes lipid accumulation object to increase by the mediation activating macrophage of TLR9
Add, the amplification of foam cells.In addition, in the fiber fat lesion of human atherosclerosis, plasmacytoid dendritic cellss
(plasmacytoid dendritic cell, pDC) also expresses TLR9.In vivo, TLR9 is activated by CpG DNA fragmentations,
It stimulates CD4+T cells to be moved to smooth muscle, promotes the secretion of interferon (IFN-α).Studies have found that, TLR9 adds in addition to having recently
The formation of fast AS patches also protects body to alleviate the effect of AS.Evidence suggests the activation stimulation cDC of TLR9 is produced in vivo
Raw interleukin (IL) -10, can inhibit pDC to express IFN-α in turn.However there is experiment in vivo to prove, in ApoE-/-Mouse
TLR9 shields in atherosclerosis in model.Christine Koulis et al. have found, biradical to TLR9, ApoE
It because of the C57BL/6 mouse feeding food rich in fat of knockout, is compared, is observed to the two atherosclerosis with the mouse of ApoE
The influence of lesion degree.The results show that TLR9 plays the role of protection in AS, this result is supported in Apo-/-In mouse
Oligodeoxynucleotide stimulates the degree of TLR9 reduction pathological lesions.TLR3 also has similar difference with TLR7, i.e., real in vitro
There is pro-inflammatory effect in testing, there is antiatherosclerosis in vivo.There are many cell type of expression TLR receptors in vivo, respectively
The specific difference of kind of cell TLR ligands and thus various cell types interactions in environment in vivo, effect are mutually supported
Disappear neutralization, may cause this internal external difference.In addition, various disease may be in the signal path in its recombination reaction
It is different.For example, according to SalagianniM et al., intervention vessel rebuilds the retardance for reducing TLR7 and TLR9, however TLR7,
The gene deletion of TLR9 results in the acceleration of atherosclerosis.Certainly, in TLR9 signal pathways, there is the thin of many steps
Section is all disputable, and how TLR9 and CpG DNA act on, transports of the TLR9 in cell, signal transduction mechanism also downstream
Nor being fully apparent from, these, which all need further to be studied, illustrates.
Recent study prompts：TLR families play a significant role in the inflammatory and immune response of coronary heart disease, are bacterium infections
The bridge formed with coronary heart disease inflammation, the effect in coronary heart disease have become new research hotspot.The discoveries such as Lan Jingsheng
The positive rate that mononuclearcell TLR2~5 is expressed in peripheral blood is proportionate with coronary risk factor integral.Shirak is
Confirm that the expression in TLR1, TLR4, TLR6 atherosclerotic plaque increases than normal person.Erridge etc. is sent out in zoopery
Existing TLRs families internal members have synergistic effect.
Invention content
In view of the defects and deficiencies of the prior art, the present invention intends to provide a kind of simple in structure, reasonable design makes
With the research method that TLR9 is easily applied to treatment coronary atherosclerosis；By detecting patients with coronary heart disease with RT-PCR
With the expression of TLR9mRNA in healthy person peripheral blood mononuclear cells, specify TLR9 and coronary heart disease and its prognosis have it is irrelevant simultaneously
Further what kind of relationship analysis is, the influence that Percutaneous coronary interventions express TLR9 is established newly for coronary heart disease treatment
Target spot；It is used in combination TLR9 antagonists and agonist to cause Human umbilical vein endothelial cells HUVEC damage models to intervene ox-LDL,
Effects of the TLR9 in ox-LDL endothelial cell injury atharosclerosis is further inquired into and to its intervention effect, to find
Intervention drug new AS makees an attempt.
To achieve the above object, the technical solution adopted by the present invention is：It is respectively from two side of human experimentation and cell experiment
It is researched and analysed in face；
The research step of the human experimentation is as follows：
1, research object and grouping：
In June, -2019 in April, 2017 heart internal medicine in hospital inpatient, person 150 is hat through clinical and angiographic diagnosis altogether
The patient of shape atherosclerotic heart disease (CAD) diagnoses according to typical clinical manifestation, Myocardial Enzymologic, electrocardiogram, hat
The indexs such as arteries and veins radiography meet diagnostic criteria of the World Health Organization (WHO) about coronary heart disease；Wherein acute myocardial infarction AMI (AMI)
Group 50；Stable angina pectoris (SA) organizes 50；Unstable angina (UA) organizes 50；Normal group：It is selected in 50 altogether
Without coronary disease patient, no the past History of Coronary Heart Disease, blood routine, blood fat, kidney function indicator are normal, without other autoimmune diseases
And infectious diseases performance；
2, blood sample collection：
Coronary heart disease those selected at PCI preoperative and postoperative (72h), no coronary disease patient any time point acquisition venous blood on an empty stomach
3ml, 1：9 sodium citrate anti-freezing；
3, Indexs measure：
3.1, cardiac ultrasonic：The each patient's preoperative and postoperative of CHD group respectively does a heart during cardiac ultrasound, and (acute ST-segment is lifted
The preoperative cardiac ultrasonic of high type myocardial infarction can not be done), no good Cardiac ultrasound of coronary disease patient；
3.2, patients with coronary heart disease it is preoperative, it is postoperative, without myocardium enzyme of coronary disease patient, CRP, BNP detect；
3.3, patients with coronary heart disease preoperative and postoperative, without coronary disease patient TLR9 detection；
3.3.1 it, detaches PBMCs and prepares all person persons of cell total rna acquisition venous blood 3ml on an empty stomach, with 1：9 citron
Sour sodium anti-freezing, it is single by density-gradient centrifugation method separation with the lymphocyte separation medium of 2ml after isometric normal saline dilution is added
A nucleus；Cell precipitation is collected, cell total rna is extracted, operates the cell total rna extraction agent box in strict accordance with Reagent Company
Specification carry out；Respectively by 1% agarose gel electrophoresis and UV detector, the quality of detection gained total serum IgE and
Concentration；
3.3.2, the synthesis RNA+DEPC water of cDNA：11 μ l, RNA amount 1-5 μ g；Oligle dT：1μl；70 DEG C of placements
5min places 5min on ice；5×RTase Buffer：4 μ l (10 × RTase Buffer, 2 μ l+2 μ l DEPC water)；Rnase
Inhibitor：1 μ l, dNTP：2 μ l, 37 DEG C of placement 5min；RTase：1 μ l, 42 DEG C of placements 70min, 70 DEG C of placement 10min；Institute
CDNA-20 DEG C freeze it is spare；
3.3.3, qRT-PCR takes 0.2ml thin-walled PCR pipes, numbers respectively；2 × qPCR TaqMix are added into each pipe
The forward and reverse primer mixture 0.5ul of each gene of 12.5ul, 10uM, corresponding each 1ul of cDNA (GAPDH is internal reference)；In one pipe not
Template is added to be used as negative control；Each pipe benefit adds water to 25ul；PCR reacts mixing, is placed in 480 fluorescence quantitative PCR instruments of Roche；
After 95 DEG C of 5min pre-degenerations, 95 DEG C of 15s, 65 DEG C of 35s (fluoroscopic examination), 40 cycles；
4, statistical analysis：
Each data are with mean ± standard deviationIt indicates, comparison among groups row homogeneity test of variance first, heterogeneity of variance,
Using non-parametric test (Kruskal Wallis test), variance uses variance analysis together, using SPSS23 statistical softwares to reality
It tests data and carries out statistical procedures；P≤0.05 is that difference is statistically significant.
The research step of the cell experiment is as follows：
1, cell culture and passage：
Conventional method is recovered the HUVEC that freezes, be added containing 10% fetal calf serum, 1 × 108UL-1 penicillin, 1 ×
The DMEM culture mediums of 108UL-1 streptomycin sulphates, 5 × 107UL-1 gentamicins, it is quiet in 37 DEG C, 5%CO2 incubators
Culture is set, by well-grown HUVEC with after 0.5% trypsin digestion 2-3min, cell suspension is made in gently piping and druming, often
All secondary cultures 2~3 times；
2, cell grouping and administration：
HUVEC damage models, pharmaceutical intervention group is caused to use TLR9's respectively using ox-LDL (final concentration of 30mgL-1)
Antagonist (IRS869) (final concentration of 1 μM) and (final concentration of 1 μM) of agonist (ODN1826) pretreatment 4h are further continued for containing
The culture medium of ox-LDL and relative medicine is cultivated；
3, MTS colorimetric determinations endothelial cell existence activity：
MTS colorimetric methods are common detection cells survival activity, the method for Screening Treatment factor concentration, and living cells can incite somebody to action
MTS is reduced to a kind of bluish violet Jia Za compounds, and dead cell then cannot, which has stronger at 490nm
Absorption peak, and this absorption peak and viable count are at preferable linear relationship；The specific method is as follows：By HUVEC single cell suspensions
96 orifice plates are inoculated in, every group sets 4 multiple holes, after each group HUVEC cells are carried out respective handling, carefully sops up former culture medium, often
MTS detection liquid (MTS is added in hole：Serum free medium=1：5) 100 μ l, and blank control is set, continue to cultivate 30-60min；With
Multi-function microplate reader measures OD value of each hole at 490nm, and calculates cells survival activity by following equation：Cells survival is lived
Property (%)=(sample well OD490 values-blank well OD490 values)/(control wells OD490 values-blank well OD490 values) × 100%；
4, living cells imaging system detection endothelial cell mitochondrial membrane potential variation：
HUVEC is inoculated in 96 orifice plate of glass bottom, each group cell carries out respective handling and JC-1 mitochondrial membrane electricity is added simultaneously
Position detection reagent (10 μ l JC-1 detection reagents are added in every 100 μ l culture mediums), 37 DEG C of incubation 15min in CO2 incubators；Root
The wavelength recommended according to specification carries out dynamic observation with the channels FITC and TRITC in living cells imaging system；JC-1 dye sheets
Reveal accumulating in mitochondria for potential dependence, in normal mitochondria, JC-1 is gathered in mitochondria base in polymer form
In matter, strong red fluorescence (Ex=585nm, Em=590nm) is sent out；Decline or funeral of the mitochondria of damage due to film potential
It loses, JC-1 can only be present in the form of monomer in endochylema, generate green fluorescence (Ex=514nm, Em=529nm)；According to face
The variation of color directly reflects that the variation of mitochondrial membrane potential, the degree of depolarization of mitochondria can also pass through red green fluorescence intensity
Ratio weigh；
5, living cells imaging system detection endothelial cell calcium ion variation：
HUVEC is inoculated in 96 orifice plate of glass bottom, each group cell carries out respective handling, and Fluo-3AM calcium ion fluorescence is added and visits
Needle (final concentration of 1 μM) is protected from light at 37 DEG C and is incubated 30min progress fluorescence probe loadings, then washed twice, added with D-Hank ' s liquid
Enter plasma-free DMEM medium to continue to be incubated 20min to ensure that Fluo-3AM is completely converted under the action of esterase in the cell
Fluo-3 generates stronger fluorescence to be trapped in intracellular and calcium binding；The excitation recommended according to specification
Wavelength 488nm, launch wavelength 525-530nm carry out dynamic observation with the channels FITC in living cells imaging system；
6, Western blot detect the expression of LOX-1, p38MAPK, pI κ B α, NF- κ Bp65, pNF- κ Bp65 albumen：
It after each group HUVEC cells are carried out respective handling, discards culture medium PBS and cleans 2 times, blot PBS, each ware addition etc.
Amount cell pyrolysis liquid moves to cell lysate in EP pipes after fully cracking, and the above operation carries out on ice.4℃、12000g
20min is centrifuged, is collected in supernatant to new EP pipes；Using albumen concentration in BCA kit measurement supernatants, remaining addition 5x loadings are slow
Fliud flushing, 100 DEG C of metal bath heating 5min keep albuminous degeneration, cooling spare.Sds page is prepared, each group takes equivalent
Protein sample carries out gel electrophoresis, and 90V voltage stabilizings electrophoresis about 30min uses 120V voltage stabilizing electricity instead after bromophenol blue enters separation gel
Swimming takes out gel until bromophenol blue leading edge is moved to gel bottom；Using half dry type transferring film, albumen in gel is transferred to nitric acid fibre
The plain film (NC films) of dimension；NC films are taken out after transferring film, 3h is closed using confining liquid room temperature, and the NC films merging use after closing is pressed
In corresponding primary antibody solution after specification dilution, 4 DEG C are slowly shaken and are incubated overnight, will after film 3 times is washed in the oscillation of TBST horizontal shakers
In the two corresponding anti-solution of the appropriate diluted horseradish peroxidase-labeled of NC films merging, it is incubated at room temperature 2h.TBST room temperatures wash film 3 times；
With 9120 Dual band IR laser imaging system Scanning Detctions of Odyssey, using relative density gray value as expressing quantity；
7, the expression of cellular immunofluorescence method testing goal albumen：
It after each group HUVEC cells are carried out respective handling, is washed 3 times with the PBS of precooling, liquid (methanol is fixed per hole:Ice
Acetic acid=3：1) 200 μ l fix 15min under the conditions of 4 DEG C, then are washed 3 times with the PBS of precooling, add and penetrate liquid (0.25% poly- second
The mixed liquor of glycol octyl phenyl ether and 5% dimethyl sulfoxide (DMSO)) 1ml, 37 DEG C of placement 5min, then washed 3 times with the PBS of precooling,
The 50 μ l of secondary antibody of FITC labels are added, 37 DEG C are incubated 1h, observation of taking pictures under fluorescence microscope；
8, ELISA method detection Endothelial cell culture supernatant MCP-1, sICAM-1, sVCAM-1 is horizontal：
After each group HUVEC cells are carried out respective handling, each group cells and supernatant, 1000rpm is taken to centrifuge 2min removals
Cell fragment, takes supernatant to carry out Indexs measure, and operating procedure is carried out in strict accordance with specification；
9, statistical analysis：
Each data are with mean ± standard deviationIt indicates, comparison among groups row homogeneity test of variance first, heterogeneity of variance,
Using non-parametric test (Kruskal Wallis test), variance uses variance analysis together, using SPSS23 statistical softwares to reality
It tests data and carries out statistical procedures；P≤0.05 is that difference is statistically significant.
The present invention has the beneficial effect that：The research side of the present invention that TLR9 is applied to treatment coronary atherosclerosis
Method；It is clear by detecting the expression of TLR9mRNA in patients with coronary heart disease and healthy person peripheral blood mononuclear cells with RT-PCR
TLR9 has what kind of relationship irrelevant step analysis of going forward side by side is with coronary heart disease and its prognosis, and Percutaneous coronary interventions are to TLR9 tables
The influence reached establishes new target spot for coronary heart disease treatment；It is used in combination TLR9 antagonists and agonist to cause human umbilical vein to ox-LDL
Endothelial cell HUVEC damage models are intervened, and are further inquired into TLR9 and are caused Atherosclerosis in ox-LDL endothelial cell injuries
Effect in change and to its intervention effect, makees an attempt to find the new intervention drugs of AS.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without having to pay creative labor, may be used also for those of ordinary skill in the art
With obtain other attached drawings according to these attached drawings.
Fig. 1 is the flow chart of human experimentation in the present invention.
Fig. 2 is the flow chart of cell experiment in the present invention.
Specific implementation mode
The present invention will be further described below with reference to the drawings.
Specific implementation mode one：
Referring to as shown in Figure 1, present embodiment the technical solution adopted is that：It is studied in terms of human experimentation
Analysis, the research step of the human experimentation are as follows：
1, research object and grouping：
In June, -2019 in April, 2017 heart internal medicine in hospital inpatient, person 150 is hat through clinical and angiographic diagnosis altogether
The patient of shape atherosclerotic heart disease (CAD) diagnoses according to typical clinical manifestation, Myocardial Enzymologic, electrocardiogram, hat
The indexs such as arteries and veins radiography meet diagnostic criteria of the World Health Organization (WHO) about coronary heart disease；Wherein acute myocardial infarction AMI (AMI)
Group 50；Stable angina pectoris (SA) organizes 50；Unstable angina (UA) organizes 50；Normal group：It is selected in 50 altogether
Without coronary disease patient, no the past History of Coronary Heart Disease, blood routine, blood fat, kidney function indicator are normal, without other autoimmune diseases
And infectious diseases performance；
2, blood sample collection：
Coronary heart disease those selected at PCI preoperative and postoperative (72h), no coronary disease patient any time point acquisition venous blood on an empty stomach
3ml, 1：9 sodium citrate anti-freezing；
3, Indexs measure：
3.1, cardiac ultrasonic：The each patient's preoperative and postoperative of CHD group respectively does a heart during cardiac ultrasound, and (acute ST-segment is lifted
The preoperative cardiac ultrasonic of high type myocardial infarction can not be done), no good Cardiac ultrasound of coronary disease patient；
3.2, patients with coronary heart disease it is preoperative, it is postoperative, without myocardium enzyme of coronary disease patient, CRP, BNP detect；
3.3, patients with coronary heart disease preoperative and postoperative, without coronary disease patient TLR9 detection；
3.3.1 it, detaches PBMCs and prepares all person persons of cell total rna acquisition venous blood 3ml on an empty stomach, with 1：9 citron
Sour sodium anti-freezing, it is single by density-gradient centrifugation method separation with the lymphocyte separation medium of 2ml after isometric normal saline dilution is added
A nucleus；Cell precipitation is collected, cell total rna is extracted, operates the cell total rna extraction agent box in strict accordance with Reagent Company
Specification carry out；Respectively by 1% agarose gel electrophoresis and UV detector, the quality of detection gained total serum IgE and
Concentration；
3.3.2, the synthesis RNA+DEPC water of cDNA：11 μ l, RNA amount 1-5 μ g；Oligle dT：1μl；70 DEG C of placements
5min places 5min on ice；5×RTase Buffer：4 μ l (10 × RTase Buffer, 2 μ l+2 μ l DEPC water)；Rnase
Inhibitor：1 μ l, dNTP：2 μ l, 37 DEG C of placement 5min；RTase：1 μ l, 42 DEG C of placements 70min, 70 DEG C of placement 10min；Institute
CDNA-20 DEG C freeze it is spare；
3.3.3, qRT-PCR takes 0.2ml thin-walled PCR pipes, numbers respectively；2 × qPCR TaqMix are added into each pipe
The forward and reverse primer mixture 0.5ul of each gene of 12.5ul, 10uM, corresponding each 1ul of cDNA (GAPDH is internal reference)；In one pipe not
Template is added to be used as negative control；Each pipe benefit adds water to 25ul；PCR reacts mixing, is placed in 480 fluorescence quantitative PCR instruments of Roche；
After 95 DEG C of 5min pre-degenerations, 95 DEG C of 15s, 65 DEG C of 35s (fluoroscopic examination), 40 cycles；
4, statistical analysis：
Each data are with mean ± standard deviationIt indicates, comparison among groups row homogeneity test of variance first, heterogeneity of variance,
Using non-parametric test (Kruskal Wallis test), variance uses variance analysis together, using SPSS23 statistical softwares to reality
It tests data and carries out statistical procedures；P≤0.05 is that difference is statistically significant.
Present embodiment has the beneficial effect that：TLR9 is applied to treatment coronary artery described in present embodiment
The research method of atherosis；By detecting TLR9mRNA in patients with coronary heart disease and healthy person peripheral blood mononuclear cells with RT-PCR
Expression, specify TLR9 and coronary heart disease and its prognosis to have irrelevant step analysis of going forward side by side be what kind of relationship, percutaneous coronary
The influence that interventional treatment expresses TLR9 establishes new target spot for coronary heart disease treatment；It is used in combination TLR9 antagonists and agonist to ox-
LDL causes Human umbilical vein endothelial cells HUVEC damage models to be intervened, and it is thin in ox-LDL injured endotheliums further to inquire into TLR9
Effect in born of the same parents' atharosclerosis and to its intervention effect, makees an attempt to find the new intervention drugs of AS.
Specific implementation mode two：
Referring to Fig. 2, present embodiment is researched and analysed in terms of cell experiment, cell experiment research step
It is rapid as follows：
1, cell culture and passage：
Conventional method is recovered the HUVEC that freezes, be added containing 10% fetal calf serum, 1 × 108UL-1 penicillin, 1 ×
The DMEM culture mediums of 108UL-1 streptomycin sulphates, 5 × 107UL-1 gentamicins, it is quiet in 37 DEG C, 5%CO2 incubators
Culture is set, by well-grown HUVEC with after 0.5% trypsin digestion 2-3min, cell suspension is made in gently piping and druming, often
All secondary cultures 2~3 times；
2, cell grouping and administration：
HUVEC damage models, pharmaceutical intervention group is caused to use TLR9's respectively using ox-LDL (final concentration of 30mgL-1)
Antagonist (IRS869) (final concentration of 1 μM) and (final concentration of 1 μM) of agonist (ODN1826) pretreatment 4h are further continued for containing
The culture medium of ox-LDL and relative medicine is cultivated；
3, MTS colorimetric determinations endothelial cell existence activity：
MTS colorimetric methods are common detection cells survival activity, the method for Screening Treatment factor concentration, and living cells can incite somebody to action
MTS is reduced to a kind of bluish violet Jia Za compounds, and dead cell then cannot, which has stronger at 490nm
Absorption peak, and this absorption peak and viable count are at preferable linear relationship；The specific method is as follows：By HUVEC single cell suspensions
96 orifice plates are inoculated in, every group sets 4 multiple holes, after each group HUVEC cells are carried out respective handling, carefully sops up former culture medium, often
MTS detection liquid (MTS is added in hole：Serum free medium=1：5) 100 μ l, and blank control is set, continue to cultivate 30-60min；With
Multi-function microplate reader measures OD value of each hole at 490nm, and calculates cells survival activity by following equation：Cells survival is lived
Property (%)=(sample well OD490 values-blank well OD490 values)/(control wells OD490 values-blank well OD490 values) × 100%；
4, living cells imaging system detection endothelial cell mitochondrial membrane potential variation：
HUVEC is inoculated in 96 orifice plate of glass bottom, each group cell carries out respective handling and JC-1 mitochondrial membrane electricity is added simultaneously
Position detection reagent (10 μ l JC-1 detection reagents are added in every 100 μ l culture mediums), 37 DEG C of incubation 15min in CO2 incubators；Root
The wavelength recommended according to specification carries out dynamic observation with the channels FITC and TRITC in living cells imaging system；JC-1 dye sheets
Reveal accumulating in mitochondria for potential dependence, in normal mitochondria, JC-1 is gathered in mitochondria base in polymer form
In matter, strong red fluorescence (Ex=585nm, Em=590nm) is sent out；Decline or funeral of the mitochondria of damage due to film potential
It loses, JC-1 can only be present in the form of monomer in endochylema, generate green fluorescence (Ex=514nm, Em=529nm)；According to face
The variation of color directly reflects that the variation of mitochondrial membrane potential, the degree of depolarization of mitochondria can also pass through red green fluorescence intensity
Ratio weigh；
5, living cells imaging system detection endothelial cell calcium ion variation：
HUVEC is inoculated in 96 orifice plate of glass bottom, each group cell carries out respective handling, and Fluo-3AM calcium ion fluorescence is added and visits
Needle (final concentration of 1 μM) is protected from light at 37 DEG C and is incubated 30min progress fluorescence probe loadings, then washed twice, added with D-Hank ' s liquid
Enter plasma-free DMEM medium to continue to be incubated 20min to ensure that Fluo-3AM is completely converted under the action of esterase in the cell
Fluo-3 generates stronger fluorescence to be trapped in intracellular and calcium binding；The excitation recommended according to specification
Wavelength 488nm, launch wavelength 525-530nm carry out dynamic observation with the channels FITC in living cells imaging system；
6, Western blot detect the expression of LOX-1, p38MAPK, pI κ B α, NF- κ Bp65, pNF- κ Bp65 albumen：
It after each group HUVEC cells are carried out respective handling, discards culture medium PBS and cleans 2 times, blot PBS, each ware addition etc.
Amount cell pyrolysis liquid moves to cell lysate in EP pipes after fully cracking, and the above operation carries out on ice.4℃、12000g
20min is centrifuged, is collected in supernatant to new EP pipes；Using albumen concentration in BCA kit measurement supernatants, remaining addition 5x loadings are slow
Fliud flushing, 100 DEG C of metal bath heating 5min keep albuminous degeneration, cooling spare.Sds page is prepared, each group takes equivalent
Protein sample carries out gel electrophoresis, and 90V voltage stabilizings electrophoresis about 30min uses 120V voltage stabilizing electricity instead after bromophenol blue enters separation gel
Swimming takes out gel until bromophenol blue leading edge is moved to gel bottom；Using half dry type transferring film, albumen in gel is transferred to nitric acid fibre
The plain film (NC films) of dimension；NC films are taken out after transferring film, 3h is closed using confining liquid room temperature, and the NC films merging use after closing is pressed
In corresponding primary antibody solution after specification dilution, 4 DEG C are slowly shaken and are incubated overnight, will after film 3 times is washed in the oscillation of TBST horizontal shakers
In the two corresponding anti-solution of the appropriate diluted horseradish peroxidase-labeled of NC films merging, it is incubated at room temperature 2h.TBST room temperatures wash film 3 times；
With 9120 Dual band IR laser imaging system Scanning Detctions of Odyssey, using relative density gray value as expressing quantity；
7, the expression of cellular immunofluorescence method testing goal albumen：
It after each group HUVEC cells are carried out respective handling, is washed 3 times with the PBS of precooling, liquid (methanol is fixed per hole:Ice
Acetic acid=3：1) 200 μ l fix 15min under the conditions of 4 DEG C, then are washed 3 times with the PBS of precooling, add and penetrate liquid (0.25% poly- second
+ 5% dimethyl sulfoxide (DMSO) mixed liquor of glycol octyl phenyl ether) 1ml, 37 DEG C of placement 5min, then washed 3 times with the PBS of precooling, it is added
The 50 μ l of secondary antibody of FITC labels, 37 DEG C are incubated 1h, observation of taking pictures under fluorescence microscope；
8, ELISA method detection Endothelial cell culture supernatant MCP-1, sICAM-1, sVCAM-1 is horizontal：
After each group HUVEC cells are carried out respective handling, each group cells and supernatant, 1000rpm is taken to centrifuge 2min removals
Cell fragment, takes supernatant to carry out Indexs measure, and operating procedure is carried out in strict accordance with specification；
9, statistical analysis：
Each data are with mean ± standard deviationIt indicates, comparison among groups row homogeneity test of variance first, heterogeneity of variance,
Using non-parametric test (Kruskal Wallis test), variance uses variance analysis together, using SPSS23 statistical softwares to reality
It tests data and carries out statistical procedures；P≤0.05 is that difference is statistically significant.
The above is merely illustrative of the technical solution of the present invention and unrestricted, and those of ordinary skill in the art are to this hair
The other modifications or equivalent replacement that bright technical solution is made, as long as it does not depart from the spirit and scope of the technical scheme of the present invention,
It is intended to be within the scope of the claims of the invention.
Claims (2)
1. a kind of research method that TLR9 is applied to treatment coronary atherosclerosis, it is characterised in that：It is from human experimentation
Aspect is researched and analysed, and the research step of the human experimentation is as follows：
(1), research object and grouping：
In June, -2019 in April, 2017 heart internal medicine in hospital inpatient, person 150 is coronal dynamic through clinical and angiographic diagnosis altogether
The cardiopathic patient of pulse atherosclerosis diagnoses according to typical clinical manifestation, Myocardial Enzymologic, electrocardiogram, coronary angiography etc.
Index meets diagnostic criteria of the World Health Organization about coronary heart disease；Wherein acute myocardial infarction AMI group 50；Stable angina pectoris
Group 50；Unstable angina group 50；Normal group：It is selected in 50 altogether without coronary disease patient, no the past History of Coronary Heart Disease, blood
Routine, blood fat, kidney function indicator are normal, no other autoimmune diseases and infectious diseases performance；
(2), blood sample collection：
Those selected is preoperative and postoperative in PCI for coronary heart disease, no coronary disease patient any time point empty stomach acquisition venous blood 3ml, and 1：9 Chinese holly
Rafter acid sodium anti-freezing；
(3), Indexs measure：
(3.1), cardiac ultrasonic：The each patient's preoperative and postoperative of CHD group is respectively a heart during cardiac ultrasound, no coronary disease patient
Cardiac ultrasound of row；
(3.2), patients with coronary heart disease it is preoperative, it is postoperative, without myocardium enzyme of coronary disease patient, CRP, BNP detect；
(3.3), patients with coronary heart disease preoperative and postoperative, without coronary disease patient TLR9 detection；
(3.3.1), it detaches PBMCs and prepares all person persons of cell total rna acquisition venous blood 3ml on an empty stomach, with 1：9 citric acid
Sodium anti-freezing, it is single by density-gradient centrifugation method separation with the lymphocyte separation medium of 2ml after isometric normal saline dilution is added
Nucleus；Collect cell precipitation, extract cell total rna, operation in strict accordance with Reagent Company cell total rna extraction agent box
Specification carries out；Respectively by 1% agarose gel electrophoresis and UV detector, the quality of detection gained total serum IgE and dense
Degree；
The synthesis RNA+DEPC water of (3.3.2), cDNA：11 μ l, RNA amount 1-5 μ g；Oligle dT：1μl；70 DEG C of placement 5min,
5min is placed on ice；5×RTase Buffer：4μl；Rnase Inhibitor：1 μ l, dNTP：2 μ l, 37 DEG C of placement 5min；
RTase：1 μ l, 42 DEG C of placements 70min, 70 DEG C of placement 10min；CDNA-20 DEG C of gained freezes spare；
(3.3.3), qRT-PCR take 0.2ml thin-walled PCR pipes, number respectively；2 × qPCR TaqMix are added into each pipe
Each gene of 12.5ul, 10uM forward and reverse primer mixture 0.5ul, corresponding each 1ul of cDNA；It is not added with template in one pipe and is used as feminine gender
Control；Each pipe benefit adds water to 25ul；PCR reacts mixing, is placed in 480 fluorescence quantitative PCR instruments of Roche；95 DEG C of 5min pre-degenerations
Afterwards, 95 DEG C of 15s, 65 DEG C of 35s, 40 cycles；
(4), statistical analysis：
Each data indicate that comparison among groups row homogeneity test of variance first, heterogeneity of variance is examined using nonparametric with mean ± standard deviation
It tests, variance uses variance analysis together, and statistical procedures are carried out to experimental data using SPSS23 statistical softwares；P≤0.05 is poor
It is different statistically significant.
2. a kind of research method that TLR9 is applied to treatment coronary atherosclerosis, it is characterised in that：It is from cell experiment
Aspect is researched and analysed, and the research step of the cell experiment is as follows：
(1), cell culture and passage：
Recover the HUVEC frozen, is added containing 10% fetal calf serum, 1 × 108UL-1 penicillin, 1 × 108UL-1 sulfate chains
The DMEM culture mediums of mycin, 5 × 107UL-1 gentamicins, the stationary culture in 37 DEG C, 5%CO2 incubators, will grow good
Good HUVEC is with after 0.5% trypsin digestion 2-3min, and cell suspension is made in gently piping and druming, weekly secondary culture 2~3
It is secondary；
(2), cell grouping and administration：
Cause HUVEC damage models, pharmaceutical intervention group that the antagonist of TLR9 and agonist is used to pre-process 4h respectively using ox-LDL,
It is further continued for being cultivated with the culture medium containing ox-LDL and relative medicine；
(3), MTS colorimetric determinations endothelial cell existence activity：
HUVEC single cell suspensions are inoculated in 96 orifice plates, every group sets 4 multiple holes, and each group HUVEC cells are carried out respective handling
Afterwards, former culture medium is carefully sopped up, MTS is added per hole and detects 100 μ l of liquid, and sets blank control, continues to cultivate 30-60min；With more
Function microplate reader measures OD value of each hole at 490nm, and calculates cells survival activity by following equation：Cells survival activity
(%)=(sample well OD490 values-blank well OD490 values)/(control wells OD490 values-blank well OD490 values) × 100%；
(4), living cells imaging system detection endothelial cell mitochondrial membrane potential variation：
HUVEC is inoculated in 96 orifice plate of glass bottom, each group cell carries out respective handling and the inspection of JC-1 mitochondrial membrane potentials is added simultaneously
Test agent, 37 DEG C of incubation 15min in CO2 incubators；Wavelength is carried out with the channels FITC and TRITC in living cells imaging system
Dynamic observation；JC-1 dyestuffs show the accumulating in mitochondria of potential dependence, and in normal mitochondria, JC-1 is with polymer
Form is gathered in mitochondrial matrix, sends out strong red fluorescence；The mitochondria of damage due to film potential decline or forfeiture,
JC-1 can only be present in the form of monomer in endochylema, generate green fluorescence；Directly reflect mitochondrial membrane according to the variation of color
The degree of depolarization of the variation of current potential, mitochondria can also be weighed by the ratio of red green fluorescence intensity；
(5), living cells imaging system detection endothelial cell calcium ion variation：
HUVEC is inoculated in 96 orifice plate of glass bottom, each group cell carries out respective handling, and Fluo-3AM calcium ion fluorescents are added,
It is protected from light at 37 DEG C and is incubated 30min progress fluorescence probe loadings, then washed twice with D-Hank ' s liquid, serum-free DMEM cultures are added
Base continues to be incubated 20min to ensure that Fluo-3AM is completely converted into Fluo-3 under the action of esterase in the cell, to be detained
In the cell stronger fluorescence is generated with calcium binding；Excitation wavelength 488nm, launch wavelength 525-530nm are logical with FITC
Road carries out dynamic observation in living cells imaging system；
(6), Western blot detect the expression of LOX-1, p38MAPK, pI κ B α, NF- κ Bp65, pNF- κ Bp65 albumen：
It after each group HUVEC cells are carried out respective handling, discards culture medium PBS and cleans 2 times, blot PBS, it is thin that equivalent is added in each ware
Cellular lysate liquid moves to cell lysate in EP pipes after fully cracking, and the above operation carries out on ice, 4 DEG C, 12000g centrifugations
20min is collected in supernatant to new EP pipes；Using albumen concentration in BCA kit measurement supernatants, remaining addition 5x loading buffers
Liquid, 100 DEG C of metal bath heating 5min keep albuminous degeneration, cooling spare；Sds page is prepared, each group takes equivalent egg
White sample carries out gel electrophoresis, and 90V voltage stabilizings electrophoresis about 30min uses 120V voltage stabilizing electrophoresis instead after bromophenol blue enters separation gel,
It is moved to gel bottom to bromophenol blue leading edge, takes out gel；Using half dry type transferring film, albumen in gel is transferred to nitrocellulose
Film；NC films are taken out after transferring film, close 3h using confining liquid room temperature, the NC films after closing are placed in and are diluted using by specification
In corresponding primary antibody solution afterwards, 4 DEG C are slowly shaken and are incubated overnight, and after film 3 times is washed in the oscillation of TBST horizontal shakers, NC films are placed in suitable
In the two corresponding anti-solution for measuring diluted horseradish peroxidase-labeled, it is incubated at room temperature 2h；TBST room temperatures wash film 3 times；Use Odyssey
9120 Dual band IR laser imaging system Scanning Detctions, using relative density gray value as expressing quantity；
(7), the expression of cellular immunofluorescence method testing goal albumen：
It after each group HUVEC cells are carried out respective handling, is washed 3 times with the PBS of precooling, liquid, methanol is fixed per hole:Glacial acetic acid
=3：1,200 μ l fix 15min under the conditions of 4 DEG C, then are washed 3 times with the PBS of precooling, add and penetrate liquid, 0.25% polyethylene glycol
The mixed liquor of octyl phenyl ether and 5% dimethyl sulfoxide (DMSO), 1ml, 37 DEG C of placement 5min, then washed 3 times with the PBS of precooling, it is added
The 50 μ l of secondary antibody of FITC labels, 37 DEG C are incubated 1h, observation of taking pictures under fluorescence microscope；
(8), ELISA method detection Endothelial cell culture supernatant MCP-1, sICAM-1, sVCAM-1 is horizontal：
After each group HUVEC cells are carried out respective handling, each group cells and supernatant, 1000rpm is taken to centrifuge 2min and remove cell
Fragment takes supernatant to carry out Indexs measure；
(9), statistical analysis：
Each data indicate that comparison among groups row homogeneity test of variance first, heterogeneity of variance is examined using nonparametric with mean ± standard deviation
It tests, variance uses variance analysis together, and statistical procedures are carried out to experimental data using SPSS23 statistical softwares；P≤0.05 is poor
It is different statistically significant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810324827.6A CN108546750A (en) | 2018-04-12 | 2018-04-12 | A kind of research method that TLR9 is applied to treatment coronary atherosclerosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810324827.6A CN108546750A (en) | 2018-04-12 | 2018-04-12 | A kind of research method that TLR9 is applied to treatment coronary atherosclerosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108546750A true CN108546750A (en) | 2018-09-18 |
Family
ID=63514647
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810324827.6A Pending CN108546750A (en) | 2018-04-12 | 2018-04-12 | A kind of research method that TLR9 is applied to treatment coronary atherosclerosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108546750A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116334214A (en) * | 2023-04-04 | 2023-06-27 | 暨南大学附属第一医院（广州华侨医院） | CERNA regulation network for biomarker for identifying acute myocardial infarction and stable angina pectoris and application thereof |
| CN116942842A (en) * | 2023-07-17 | 2023-10-27 | 无锡市第二人民医院 | Nano silver bonded with cytoblast promoting functional nucleic acid, and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103558392A (en) * | 2013-10-17 | 2014-02-05 | 中国中医科学院中药研究所 | Anti-atherosclerosis drug efficacy evaluation method based on inflammatory responses |
-
2018
- 2018-04-12 CN CN201810324827.6A patent/CN108546750A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103558392A (en) * | 2013-10-17 | 2014-02-05 | 中国中医科学院中药研究所 | Anti-atherosclerosis drug efficacy evaluation method based on inflammatory responses |
Non-Patent Citations (6)
| Title |
|---|
| 张开滋等: "《临床心力衰竭学》", 31 October 2014, 湖南科学技术出版社 * |
| 方石虎: "冠心病患者全血中miR-126表达的临床意义", 《临床和实验医学杂志》 * |
| 陈为等: "OX-LDL介导内皮细胞损伤及其对TLR-4和EpCAM表达的影响", 《吉林大学学报（医学版）》 * |
| 陈宁等: "《全科医生临床诊疗手册》", 30 June 2016, 中国医药科技出版社 * |
| 陈芳等: "《慢性咳嗽中西医诊治名老中医王会仍临床经验》", 31 March 2015, 中国医药科技出版社 * |
| 马春梅: "APOE-/-小鼠TLR9介导巨噬细胞极化效应对动脉粥样硬化作用的研究", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116334214A (en) * | 2023-04-04 | 2023-06-27 | 暨南大学附属第一医院（广州华侨医院） | CERNA regulation network for biomarker for identifying acute myocardial infarction and stable angina pectoris and application thereof |
| CN116942842A (en) * | 2023-07-17 | 2023-10-27 | 无锡市第二人民医院 | Nano silver bonded with cytoblast promoting functional nucleic acid, and preparation method and application thereof |
| CN116942842B (en) * | 2023-07-17 | 2024-05-07 | 无锡市第二人民医院 | Nano silver bonded with cytoblast promoting functional nucleic acid, and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bascones et al. | Apoptosis and cell cycle arrest in oral lichen planus: hypothesis on their possible influence on its malignant transformation | |
| Tian et al. | Depletion of energy reserve via the creatine kinase reaction during the evolution of heart failure in cardiomyopathic hamsters | |
| CN1954825B (en) | Supermicro Tongxinluo Chinese herbal composite and its new usage | |
| CN105349642B (en) | A kind of acute myocardial infarction AMI marker and its application | |
| CN106399309B (en) | A kind of long non-coding RNA and its application in diagnosis/treatment T2DM | |
| CN108841965A (en) | Application of the TLCD1 in the diagnosis of Metastasis in Breast Cancer, prognosis and treatment | |
| Yao et al. | Combination of HGF and IGF-1 promotes connexin 43 expression and improves ventricular arrhythmia after myocardial infarction through activating the MAPK/ERK and MAPK/p38 signaling pathways in a rat model | |
| CN108546750A (en) | A kind of research method that TLR9 is applied to treatment coronary atherosclerosis | |
| Ding et al. | Parthenolide ameliorates neurological deficits and neuroinflammation in mice with traumatic brain injury by suppressing STAT3/NF-κB and inflammasome activation | |
| Chen et al. | STING inhibition accelerates the bone healing process while enhancing type H vessel formation | |
| Quan et al. | Thymosin β4 promotes the survival and angiogenesis of transplanted endothelial progenitor cells in the infarcted myocardium | |
| US9499851B2 (en) | Wound healing metakaryotic stem cells and methods of use thereof | |
| CN105349641A (en) | Acute myocardial infarction related gene SERPINB13 and application thereof | |
| CN108929903A (en) | Marker and kit for chronic periodontitis screening, diagnosis, therapeutic evaluation | |
| CN106701902A (en) | FOXR2 gene and application of expression product to diagnosis and treatment of liver cancer | |
| CN112557658B (en) | Application of neutrophil elastase in preparation of product for diagnosing biliary tract occlusion | |
| CN109097358A (en) | A kind of lncRNA is preventing or is treating the application in hypertension | |
| CN107012207A (en) | LRP5 is used as clinical diagnosis and the application of suppression digestive system tumor | |
| RU2453845C1 (en) | Method for assessing status of blood vessel wall | |
| CN104258377B (en) | Application of the PIK3C2A albumen in liver-cancer medicine is treated | |
| CN105920025B (en) | Topiramate is applied in the drug for the treatment of myocardial infarction | |
| Jacobse et al. | Eosinophils exert antitumorigenic effects in the development of esophageal squamous cell carcinoma | |
| CN111474364A (en) | Use of human RAB22A and related products | |
| CN107217055B (en) | A kind of cancer diagnosis chip and its kit | |
| RU2486516C1 (en) | Diagnostic technique for stage of chronic hepatitis c in rna-hcv "+" patients |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180918 |