CN106018824B - Purposes of the aryl hydrocarbon receptor in drug-induced heart disease biomarker is prepared - Google Patents
Purposes of the aryl hydrocarbon receptor in drug-induced heart disease biomarker is prepared Download PDFInfo
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- CN106018824B CN106018824B CN201610332687.8A CN201610332687A CN106018824B CN 106018824 B CN106018824 B CN 106018824B CN 201610332687 A CN201610332687 A CN 201610332687A CN 106018824 B CN106018824 B CN 106018824B
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- myocardial damage
- triptolide
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- 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
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
Abstract
The invention belongs to biological technical field, purposes of more particularly to a kind of aryl hydrocarbon receptor in drug-induced heart disease biomarker is prepared.
Description
Technical field
The invention belongs to biological technical field, more particularly to aryl hydrocarbon receptor is preparing drug-induced heart disease biological marker
Purposes in thing.
Background technology
In broad sense, the heart disease caused by all myocardium toxicities direct or indirect medicine, either because of medicine
To the abnormal or various arrhythmia cordis of myocardium depolarization and multipole caused by the influence of cardiac electrophysiology, or cardiac muscle shrinkage because
The toxic action of medicine and be suppressed, and then the heart failure for inducing or aggravating, or because causing the heart to certain drug allergy
Myositis, pericarditis, can be referred to as drug-induced heart disease (drug-induced heart diseases).With other drug induccds
Disease is the same, and drug-induced heart disease can in itself, due to the doctor of medication and the aspect of patient three controlled be facilitated medicine,
But usual medicine is the leading factor for causing lesion with drug interaction in itself.Medicine involves heart, and person is mainly to cardiac muscle
Infringement, but a small number of medicines also involve pericardium and the internal membrane of heart.
Tripterygium wilfordii (Tripterygium wilfrdii Hook.f.) also known as herba fibraureae recisae, herba fibraureae recisae wood, allusion quotation wax rattan, gelsemium elegan etc.,
Belong to Wei Mao sections bejuco.Its English name：Common Threewingnut Root, latin name：Radix
Tripterygii Wilfordii Tripterygium wilfordii HooK.f.Documents and materials record its bitter, pungent, property
Cool, big poison returns liver and kidney channel.The effects such as detoxifying with dispelling wind and eliminating dampness, promoting blood circulation and removing obstruction in channels, swelling and pain relieving, desinsection.The root of tripterygium wilfordii,
Neck, Ye Junke are as medicinal, and main medicinal part is its root.Found in clinical practice, Tripterygium Preparations treatment rheumatoid arthrosis
Scorching mechanism is similar to corticoid, but the side effect without steroids, and better tolerance, potential applicability in clinical practice is wide.But
Research in recent years finds that tripterygium wilfordii can cause toxic myocarditis, cause cardiac conduction obstacle, and severe patient may occur in which cardiogenic
Shock and heart failure.In acute toxic cardiac damage caused by various Tripterygium Preparations, and caused with triptolide
Cardiac damage it is the most serious.
Some researchs show that triptolide has different inhibitory action, such as increase mitochondrial oxidation stress, P- sugar egg
The expression of white gene and function, Nuclear factor kappa B (NF- κ B) signals and cyclooxygenase-2 expression, TAK1/MAP3K7 activity is (by dry
Disturb the formation of the complicated things of TAK1/MAP3K7--TAB1/MAP3K7IP1), lower Rac1 and JAK/STAT3 paths, and the oxygen induced
Change stress.In addition, some research groups report that triptolide is the suppression of transcription factor II (TFIIH) human-like rna plymerase ii
Preparation.Report that triptolide increases oxidative stress and inducing cell apoptosis and subsequent by mitochondria pathway and goes to pole week et al.
Change mitochondrial membrane potential, reduce Bax/Bcl-2 ratios, release cromoci and activation Caspase-3.Some study channel syndrome
Real, in non-myocardial infarction system, tripterygium wilfordii toxicity is related to mitochondria pathway.A nearest research shows, tripterygium wilfordii first
Element suppresses transcription and the Nucleotide Sequence Analysis of rna plymerase ii mediation by transcription factor TFIIH.Gene delection or Thunder God
Rattan A prime, which suppresses TFIIH, can induce the Apoptosis that the tumour cell JNK of p53 defects is relied on.
Biomarker (Biomarker) refers to can be with Mk system, organ, tissue, cell and subcellular structure or work(
The biochemical indicator of change or the change that may occur of energy, with purposes widely.Biomarker is examined available for disease
Break, judge staging or for evaluating the safety and efficacy of new drug or new treatment in target group.It is clinical at present
The biochemical indicator of upper diagnosing cardiac has been widely used in the detection of cardiotoxicity of medicine.Wherein, cardiac muscle troponin I
(cTnI) it is the albumen existed only in ventricular muscles and heart muscle specific to a kind of cardiac muscle cell.CTnI molecular weight is
22ku, serum half-life is 2h or so.CTn I there are about 3%~6% in cardiac muscle and be present in a free form in endochylema, remaining
Part is then present in muscle fibril with combining form.CTn I content is seldom in normal human serum, in acute myocardial infarction
(AMI) after falling ill, serum cTn I start rise more than 3~6h, and 10~24h reaches peak, and 5~7d recovers normal.With myocardium creatine
Kinases MB (CK-MB) is compared, and cTn I contents in cardiac muscle are more, and molecular weight is less than normal, is easily tested after myocardial damage in serum
Go out, so it is more sensitive, the microlesion available for detection cardiac muscle.In addition, there is particle in the myocardium infiltrating cells of document report
Enzyme B expression quantity and the cardiac damage order of severity are into positive correlation.
Myocardial enzymes include creatine kinase isozyme (CK-MB), lactic dehydrogenase (LDH) etc., are commonly used to the joint-detection heart
Whether flesh is damaged.
Myocardium CK-MB (CK-MB)：Detect that CK-MB hypotypes are used for the diagnosis of acute myocardial infarction AMI (AMI),
CK-MB has 3 kinds of hypotypes to be present in vivo, and CK-MB1 is blood plasma type, and CK-MB2 is tectotype, is present in cardiac muscle cell, takes care
Blood is released into during injury of muscle and is changed into CK-MB1.Under normal circumstances, CK-MB2/CK-MB1 ＜ 1.0,1.5 are risen to such as ratio~
When 1.7 there is myocardial damage in prompting, and detection CK-MB1 and CK-MB2 hypotypes more have Sensitivity and Specificity to diagnosis AMI.CK-MB
Many to play 3~6h rises after being ill in AMI, 12~24h reaches peak, and 2~3d recovers normal.CK-MB rises are that the sensitivity of AMI early stages refers to
One of mark.
Catalase (CAT) is a kind of enzyme scavenger, also known as catalase, is the desmoenzyme using ferriporphyrin as prothetic group.
It can promote H2O2Molecular oxygen and water are decomposed into, internal hydrogen peroxide is removed, so that cell protects against H2O2Murder by poisoning,
It is one of key enzyme of biophylaxis system.
Aryl hydrocarbon receptor (AHR) is the orphan nuclear receptor of a mediation xenobiotic metabolism, in the hair of cardiovascular system
Educate and functionally there is key effect.Hypoxemia, endothelin-1 expression increase, systemic hypertension, the heart is presented in AHR loss
Flesh plumpness and fibrosis.There are some researches show lacking AHR in vivo causes left ventricular function to be remarkably decreased, and adriamycin processing promotes the heart
Dirty p53 gene activations and apoptosis increase.Except the dioxin toxic action of mediation, some researchs show that AHR also assists in human body disease
The effect of sick system of defense.Some datas show, exposed to pollutant, such as PCB, B [a] P, TBT and smoke from cigarette increase
AHR is expressed.However, AHR expression increases are but closely related with allergy.
There are some researches show systemic hypertension caused by AHR missings and the expression of increase endothelin -1.It is another to there is research to report
Road, the expression for lacking AHR promotes the α 1D adrenocepters of blood vessel and strengthens the effect of Angiotensin II.Several groups of accounts
Bright, the missing of AHR genes causes cardiomegaly.On the contrary, glucose intake can dramatically increase heart AHR and Nrf2 activity, improve
The adjoint heart failure of spontaneous hypertensive rat.
The content of the invention
The purpose of the present invention aims to provide purposes of the AHR in drug-induced heart disease biomarker is prepared.
Specifically, the first aspect of the present invention there is provided aryl hydrocarbon receptor and be carried in preparation detection tripterygium wilfordii or tripterygium wilfordii
The application in the biomarker of myocardial damage caused by thing is taken, and the tripterygium wilfordii or triperygium wilfordii extractive are with triptolide
For active component.
In a preference, the degree of the myocardial damage will not cause myocardium LDH, cardiac muscle CK-MB or cardiac muscle CAT work
Property reduction.
In another preference, the degree of the myocardial damage will not cause the change in concentration of blood plasma granzyme B.
In another preference, the degree of the myocardial damage will not cause the concentration of blood plasma Cardiac Troponin-I to become
Change.
The second aspect of the present invention there is provided aryl hydrocarbon receptor myocardial damage caused by detection triptolide is prepared
Biomarker in application.
In a preference, the degree of the myocardial damage will not cause myocardium LDH, cardiac muscle CK-MB or cardiac muscle CAT work
Property reduction.
In another preference, the degree of the myocardial damage will not cause the change in concentration of blood plasma granzyme B.
In another preference, the degree of the myocardial damage will not cause the concentration of blood plasma Cardiac Troponin-I to become
Change.
The details of various aspects of the present invention will be able to detailed description in subsequent chapters and sections.By hereafter and claim
Description, the features of the present invention, purpose and advantage will become apparent from.
Brief description of the drawings
Fig. 1 is the metamorphosis (HE dyeing) of rat heart muscle tissue after 14 days once to rat triptolide 1mg/kg
Fig. 2 detects once to give rat triptolide 1mg/kg after 14 days with LDH and CK-BM and with CAT kits
LDH, CK-BM and CAT activity in heart
Fig. 3 uses Cardiac Troponin-I kit once to give rat triptolide 1mg/kg, after 14 days and big respectively
The concentration of mouse granzyme B (GzmB) kit measurement Cardiac Troponin-I and rat granzyme B
Fig. 4 uses rat aryl hydrocarbon receptor kit measurement blood plasma once to give rat triptolide 1mg/kg after 14 days
With the concentration of AHR in cardiac muscle
Fig. 5 is once gives rat triptolide 1mg/kg, and real-time quantitative RT-PCR is determined in rat heart muscle after 14 days
A kind of CYP1A1 (p450 enzymes by AHR regulating and expressings) expression
Fig. 6 is the form of rat liver (A) and kidney (B) tissue after 14 days once to rat triptolide 1mg/kg
Change (HE dyeing)
Fig. 7 is once gives rat triptolide 1mg/kg, with LDH and CK-BM kits detection liver (A, C) after 14 days
With kidney (B, D) interior LDH and CK-BM activity
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.Reagent and raw material used in the following example can be obtained by commercial sources purchase
.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition or according to proposed by manufacturer
Condition.Unless otherwise defined, all specialties used in text known to scientific words and one skilled in the art with anticipating
Justice is identical.In addition, any method similar or impartial to described content and material all can be applied in the inventive method.Wen Zhong
Described preferable implementation only presents a demonstration with material to be used.
The features described above that the present invention is mentioned, or the feature that embodiment is mentioned can be in any combination.Patent specification is taken off
All features shown can be used in combination with any combinations thing form, and each feature disclosed in specification can provide phase with any
The alternative characteristics substitution of same, impartial or similar purpose.Therefore except there is special instruction, disclosed feature is only impartial or similar
The general example of feature.
Toxic action of the triptolide of embodiment 1 to rat heart
1.1 purpose：Observe once heavy dose of Cardiotoxicity to triptolide.
1.2 experiment material：
1.2.1 reagent：CMC-Na is purchased from the good medicine of Shanghai section purchased from Chemical Reagent Co., Ltd., Sinopharm Group, triptolide
Examine equipment Co., Ltd.
1.2.2 experimental animal and feed：SD rats, male, body weight (120g~140g), of the right age, health.Purchased from Shang Haixi
Pul-Bi Kai experimental animals Co., Ltd, animal credit number：SYXK (Shanghai) 2013-0058.Animal feed irradiation mouse material source
In Shanghai Shi Lin bio tech ltd, Feed Manufacturing licensing：Shanghai, which is raised, examines (2008) 04031.
1.2.3 laboratory apparatus：Superspeed refrigerated centrifuge Centrifuge 5810R are purchased from Eppendorf companies of Germany
1.3 experimental method：
SD rats are randomly divided into control group and experimental group (n=8), triptolide 1mg/kg (0.5% is disposably given
CMC-Na be solvent).The change of the death rate of daily observation animal, signs of toxicity, food consumption, chaeta outward appearance and behavior.
Dissected after 14 days, take blood and isolating cardiac, liver, kidney and other organs.Use 3.8% sodium citrate (1：9 use) make
For blood anticoagulant.Blood is centrifuged 10 minutes under 4 DEG C and 1000 × g, is obtained after blood plasma, and packing is stored in -80 DEG C of refrigerators
It is stand-by.The heart, liver, nephridial tissue sections stained with hematoxylin and Yihong (H＆E) dyeing.
1.4 experimental result：
Histotomy result shows that compared with the control, triptolide causes Cardiomyocytes Hypertrophy, oedema and part
Dissolving, space between cells increase, cardiac muscle fibre fracture, wherein male rat is even more serious than the myocardial damage of female rats (Fig. 1),
And under identical test dosage, no abnormal change is in liver and kidney (Fig. 6).
1.5 conclusion：Once heavy dose can cause myocardiac histology lesion to triptolide.There is sex in this toxicity
Difference.
Enzyme activities in cardiac muscle caused by the myocardial damage of embodiment 2
2.1 purpose：In the case where once heavy dose gives triptolide, observation myocardial damage causes enzymatic activity in cardiac muscle
Change.2.2 experiment material：
2.2.1 reagent：It is rat creatine kinase isozyme (CK-MB) ELISA kit, rat lactic dehydrogenase (LDH), big
Mouse catalase (CAT) detection kit builds up Bioengineering Research Institute's purchase from Nanjing
2.2.2 experimental animal and feed：Be the same as Example 1.2
2.2.3 laboratory apparatus：Superspeed refrigerated centrifuge Centrifuge 5810R are purchased from Eppendorf companies of Germany；Enzyme
Mark the Molecular Devices companies that instrument light absorbs ELIASA SpectraMax 190 is purchased from the U.S.；Full-automatic sample is quick
Beveller is purchased from Shanghai Jing Xin Industrial Co., Ltd.s；Ultrasonic cell disruptor is purchased from the new limited public affairs of sesame biotechnology share in Ningbo
Department
2.3 experimental method
2.3.1 zoopery：Be the same as Example 1.3
2.3.2 tissue homogenate is prepared and enzyme assay
Core, liver and nephridial tissue sample, with 1:The tissue weight (g) of 9 ratios：Physiological saline (ml) in beveller (
Under liquid nitrogen cooling) grind, setpoint frequency 75HZ continues 50 seconds, grinds 2 times, takes out tissue fluid.Ultrasound 5 times, every time 2 seconds again.So
Afterwards, centrifuge 10 minutes at 4 DEG C, 2500 revs/min, take supernatant, dilution and dispense, 10% cardiac muscular tissue's liquid is put in -80 degree ice
Case is preserved.Tissue fluid protein concentration is surveyed with Coomassie Brilliant Blue, it is rat creatine kinase isozyme (CK-MB) ELISA kit, big
Mouse lactic dehydrogenase (LDH), the enzyme assay of rat catalase (CAT) are entered according to kit manufacturer designation method
OK.
2.3.3 data processing and statistical analysis
Data processing is carried out using the softwares of GraphPad Prism 5 or SPSS15.0 softwares, data are with mean ± standard deviation
(mean ± SD) is represented, is compared to examine using pairing t- between group and is compared in progress statistical analysis, group using single factor test variance point
Analysis method (one-way ANOVA) is analyzed and processed, and p ﹤ 0.05 think there is significant difference, with statistical significance.
2.4 experimental result：
Under to triptolide 1mg/kg test doses, compared with control group, myocardium of male rat LDH, CK-MB, CAT
Active substantially reduction, and there are no significant the change of the index of female rats, this result are consistent with 1.4 HE coloration results, explanation
Female rats myocardial damage is not serious (Fig. 2).In addition, the change of the liver or kidney LDH and CK-MB of male and female rat is not obvious,
This result is consistent with 1.4 HE coloration results, illustrates the liver or kidney of male and female rat without substantially damage (Fig. 7).
2.5 conclusion：It is once heavy dose of to give triptolide, only when cardiac muscle is by major injury, damage markers
LDH, CK-MB, CAT activity can just show significant change.There are gender differences in the testing result of these toxicity indexs.
Blood plasma Cardiac Troponin-I (cTnI) and rat granzyme B (GzmB) concentration caused by the myocardial damage of embodiment 3
Change
3.1 purpose：In the case where once heavy dose gives triptolide, observation myocardial damage biomarker-cardiac muscle
The change of troponin-i and rat granzyme B (GzmB).
3.2 experiment material：
3.2.1 reagent：Rat heart muscle troponin-i enzyme-linked immunosorbent assay kit diagnoses public purchased from U.S.'s life
Take charge of (LifeDiagnostics, Inc.).Rat granzyme B (GzmB) ELISA kit is purchased from Wuhan Yi Lairuite biotechnologies
Co., Ltd
3.2.2 experimental animal and feed：Be the same as Example 1.2
3.2.3 laboratory apparatus：Superspeed refrigerated centrifuge Centrifuge 5810R are purchased from Eppendorf companies of Germany；Enzyme
Mark the Molecular Devices companies that instrument light absorbs ELIASA SpectraMax 190 is purchased from the U.S.
3.3 experimental method
3.3.1 zoopery：Be the same as Example 1.3
3.3.2 blood plasma Cardiac Troponin-I concentration mensuration
The operating procedure that the test of blood plasma Myocardial troponin-i is specified according to production company, in 96 orifice plates, respectively
Addition standard items or the μ L of sample diluting liquid 200, cover film, 25 DEG C are incubated 2 hours.Liquid in hole is discarded, washs 5 times, blots
Per hole raffinate, add after the anti-μ L of cTnl antibody diluents 100, add HRP enzyme enzyme conjugates working solutions 100ml, 25 DEG C of incubations 1 are small
When.Washing 5 times, is blotted, and is added per hole after zymolyte TMB100 μ L, overlay film, after 25 DEG C are incubated 20 minutes, adds the μ of terminate liquid 100
L, terminating reaction.ELIASA is used immediately in the OD values in each hole of 450nm wavelength measurements.
3.3.3 blood plasma rat granzyme B (GzmB) concentration mensuration
The maneuvering sequence specified according to production company, in 96 orifice plates, is separately added into standard items or the μ of sample diluting liquid 100
L, each hole is separately added into standard items or the μ L of testing sample 100, cover film, and 37 DEG C are incubated 90 minutes.Liquid is discarded, is added biological
The μ L of elementization antibody working solution 100,37 DEG C incubate 1 hour.Liquid in hole is discarded, washs 3 times, blots raffinate.Add enzyme conjugates
The μ L of working solution 100,37 DEG C incubate 30 minutes.Lucifuge at zymolyte TMB solution 90 μ L, 37 DEG C is added per hole to be incubated 15 minutes or so
Afterwards, the μ L of terminate liquid 50, terminating reaction are added.With 450nm wavelength measurement OD values.
3.3.4 data processing and statistical analysis
Data processing is carried out using the softwares of GraphPad Prism 5 or SPSS15.0 softwares, data are with mean ± standard deviation
(mean ± SD) is represented, is compared to examine using pairing t- between group and is compared in progress statistical analysis, group using single factor test variance point
Analysis method (one-way ANOVA) is analyzed and processed, and p ﹤ 0.05 think there is significant difference, with statistical significance.
3.4 experimental result：
Under to triptolide 1mg/kg test doses, detection blood plasma cTnI and GzmB concentration.Compared with control group,
Rat GzmB there are no significant change (Fig. 3 B).Only male rat blood plasma cTnI concentration shows to dramatically increase, and female is big
There was no significant difference (Fig. 3 A) by cTnI in mouse blood plasma.
3.5 conclusion：Once heavy dose of to give after triptolide, the concentration of blood plasma rat granzyme B (GzmB) is damaged to cardiac muscle
The susceptibility of wound is not obvious, and blood plasma Cardiac Troponin-I level has gender differences.
Blood plasma and the interior AHR levels change of cardiac muscle caused by the myocardial damage of embodiment 4
4.1 purpose：In the case where once heavy dose gives triptolide, blood plasma and cardiac muscle caused by observation myocardial damage
Interior AHR protein levels change.
4.2 experiment material：
4.2.1 reagent：Rat aryl hydrocarbon receptor ELISA kit is purchased from the gold medal Science and Technology Ltd. of Beijing equation one hundred
4.2.2 experimental animal and feed：Be the same as Example 1.2
4.2.3 laboratory apparatus：Superspeed refrigerated centrifuge Centrifuge 5810R are purchased from Eppendorf companies of Germany；Enzyme
Mark the Molecular Devices companies that instrument light absorbs ELIASA SpectraMax 190 is purchased from the U.S.；Full-automatic sample is quick
Beveller is purchased from Shanghai Jing Xin Industrial Co., Ltd.s；Ultrasonic cell disruptor is purchased from the new limited public affairs of sesame biotechnology share in Ningbo
Department
4.3 experimental method
4.3.1 zoopery：Be the same as Example 1.3
4.3.2 it is prepared by tissue homogenate
Core dirty tissue specimen, with 1:The tissue weight (g) of 9 ratios:PBS (PH7.4) (ml) is in beveller (in liquid nitrogen
Under cooling) grind, setpoint frequency 75HZ continues 50 seconds, grinds 2 times, takes out tissue fluid.Ultrasound 5 times, every time 2 seconds again.Then, 4
Centrifuged 20 minutes at DEG C, 2500 revs/min, take supernatant, dispensed, 10% cardiac muscular tissue's liquid is put in into -80 degree refrigerators preserves.With examining
Mas bright blue method surveys tissue fluid protein concentration.
4.3.3AHR concentration mensuration
The operating procedure specified according to production company, set respectively blank well (blank control wells are not added with sample and enzyme marking reagent,
Remaining each step operation is identical), gauge orifice, testing sample hole.Each hole is accurately loaded 50 μ l, with the rearmounted 37 DEG C of incubations of shrouding film shrouding
30 minutes.By 20 times of concentrated cleaning solutions with standby after the dilution of 20 times of distilled water.Liquid is discarded, is dried, cleaning solution is filled it up with per hole, it is quiet
Discard, be so repeated 5 times after putting 30 seconds, pat dry.Added per hole except the μ l of enzyme marking reagent 50, blank well, 37 DEG C incubate 30 minutes.
Liquid is discarded, is dried, is washed 5 times.Developer A50 μ l are first added per hole, developer B50 μ l are added, gently concussion is mixed, 37
DEG C lucifuge develops the color 10 minutes.Add the μ l of terminate liquid 50, terminating reaction per hole (now blueness is vertical turns yellow).Returned to zero with blank well,
450nm wavelength sequentially measures the OD values in each hole.
4.3.3 data processing and statistical analysis
Data processing is carried out using the softwares of GraphPad Prism 5 or SPSS15.0 softwares, data are with mean ± standard deviation
(mean ± SD) is represented, is compared to examine using pairing t- between group and is compared in progress statistical analysis, group using single factor test variance point
Analysis method (one-way ANOVA) is analyzed and processed, and p ﹤ 0.05 think there is significant difference, with statistical significance.
4.4 experimental result：
Fig. 4 is the AHR concentration in detection blood plasma and cardiac muscle under to triptolide 1mg/kg test doses.With compareing
Group is compared, and AHR protein levels have conspicuousness decline in rat heart and blood.And 2.4 results show liver kidney without obvious damage, institute
Liver kidney is not derived from AHR, illustrates that cardiac toxic caused by triptolide can make AHR protein levels in heart and blood
There is conspicuousness decline.
4.5 conclusion：Aryl hydrocarbon receptor AHR can be used as the detection cardiopathic biomarker of tripterygium wilfordii property.
CYP1A1 expression changes in the application real-time quantitative RT-PCR detection cardiac muscle of embodiment 5.
5.1 purpose：AHR downstream gene CYP1A1 expression in detection cardiac muscle, to judge that can AHR as tripterygium wilfordii first
The early monitoring and evaluation index of plain cardiac toxic
5.2 experiment material：
5.2.1 experimental animal and feed：Be the same as Example 1.2
5.2.2 reagent：Maxima SYBR Green qPCR Master Mix, Invitrogen Trizol reagents and
High-Capacity cDNA Reverse Transcription Kit are purchased from purchased from U.S. life technology (Life
Technologies) company；Gene-specific primer is synthesized by Shanghai JaRa bio-engineering corporation.
5.2.3 laboratory apparatus：The quick beveller of full-automatic sample is purchased from Shanghai Jing Xin Industrial Co., Ltd.s；Supersonic cell
Pulverizer is purchased from NingBo XinZhi Biology Science Co., Ltd；Superspeed refrigerated centrifuge Centrifuge 5810R are purchased from Germany
Eppendorf companies；PCR reverse transcriptions instrument (thermocycler, German Biometra companies)；ABI 7500fast PCR are expanded
Instrument.
5.3 experimental method：
5.3.1 zoopery：Be the same as Example 1.3
5.3.2 real-time quantitative PCR
5.3.2.1 Total RNAs extraction
5.3.2.1.1 the step of extracting cardiac muscular tissue's total serum IgE is：
1) cardiac muscular tissue 30-40mg is weighed to be put in 2ml EP pipes, plus 2 steel balls, 1ml Trizol reagents are added, are put
In tissue grinder instrument, liquid nitrogen is added in beveller, is ground, steel ball is taken out.Ultrasound for homogenization, is spaced 2s ultrasound 4s, each sample
This super 4-5 times, it is stored at room temperature 5min.Centrifuge (4 DEG C, 12000rpm, 10min), take supernatant.
2) often pipe adds 0.2mL chloroforms, fully vibrates 15s, is stored at room temperature 15min；
3) be divided into three layers after centrifugation (4 DEG C, 12000rpm, 15min), about 450 μ L upper strata aqueous phases of transfer to new 1.5mL from
In heart pipe, and isometric isopropanol is added, for several times, room temperature places 15min for reverse mixing；
4) (4 DEG C, 12000rpm, 10min) is centrifuged, abandons supernatant, it is seen that ttom of pipe white precipitate；
5) add 1mL75% ethanol washed once, 4 DEG C, 12000rpm centrifugation 5min abandon supernatant；
6) 1mL absolute ethyl alcohols are added to washed once, is abandoned after centrifugation and 10min is dried in supernatant, super-clean bench；
7) the treated water dissolving precipitations of about 15-25 μ L DEPC are added；
8) after 55 DEG C of water-bath 5min, pipette tips blow and beat mixing sample repeatedly, therefrom draw 1 μ L and are diluted to 100 μ L, ultraviolet determination
RNA concentration.
5.3.2.2 reverse transcription synthesizes cDNA
Illustrate according to reverse transcription reagent box, the total serum IgE of extraction is subjected to reverse transcription.
Each reagent after being thawed on ice, and the reverse transcription system on ice as shown in table 1 is operated：
The reverse transcription system of table 1
After each reaction solution and sample are mixed, bubble is removed in centrifugation.Put samples into PCR instrument, reverse transcription reaction program is set
For 25 DEG C × 10min, 37 DEG C × 2h, 85 DEG C × 5min, 16 DEG C × 24h.The cDNA that reverse transcription is obtained is placed in -80 DEG C of preservations.
5.3.2.3PCR amplification
According to specification, PCR reaction systems are as shown in table 2：
The PCR reaction systems of table 2
It is put into after sample blending centrifugation in PCR instrument, response procedures are：95 DEG C × 30s carries out pre-degeneration, then two-step method
(95 DEG C × 3s → 60 DEG C × 30s, 40 circulations) are expanded.As a result the Ct of target gene is entered into rower with GAPDH Ct values
Standardization, gene expression relative quantity is calculated according to comparison method, is changed using the formula 2- Δ Δs Ct relative folds for calculating gene expression.
Primer sequence see the table below：
The quantitative PCR of table 3 determines primer
5.3.3 data processing and statistical analysis
Data processing is carried out using the softwares of GraphPad Prism 5 or SPSS15.0 softwares, data are with mean ± standard deviation
(mean ± SD) is represented, is compared to examine using pairing t- between group and is compared in progress statistical analysis, group using single factor test variance point
Analysis method (one-way ANOVA) is analyzed and processed, and p ﹤ 0.05 think there is significant difference, with statistical significance.
5.4 experimental result：
Fig. 5 is that under to triptolide 1mg/kg test doses, AHR downstreams in cardiac muscle are detected with real-time quantitative RT-PCR
The mRNA expression of Gene CYP1A1, data represent the mean+SD of at least three independent experiments.
5.5 brief summary：
To under triptolide 1mg/kg test doses, existed by the AHR p450 enzyme subtype Cs YP1A1 regulated and controled mRNA expression
Substantially reduced in male and female rat heart, this is consistent with the conclusion in 4.5.So, AHR can be tripterygium wilfordii property heart disease
Diagnosis provides selection.
Many aspects involved in the present invention have been explained as above.However, it should be understood that without departing from spirit of the invention
Under the premise of, those skilled in the art can carry out equivalent change and modification to it, and the change and modification equally fall into the application
The coverage of appended claims.
Claims (8)
1. aryl hydrocarbon receptor is in the biomarker of myocardial damage caused by preparing detection tripterygium wilfordii or triperygium wilfordii extractive
Using, and the tripterygium wilfordii or triperygium wilfordii extractive are using triptolide as active component.
2. application as claimed in claim 1, it is characterised in that the degree of the myocardial damage will not cause myocardium LDH, cardiac muscle
CK-MB or cardiac muscle CAT activity reduction.
3. application as claimed in claim 1, it is characterised in that the degree of the myocardial damage will not cause blood plasma granzyme B
Change in concentration.
4. application as claimed in claim 1, it is characterised in that the degree of the myocardial damage will not cause blood plasma myocardium myo calcium
Albumen-I change in concentration.
5. application of the aryl hydrocarbon receptor in the biomarker of myocardial damage caused by preparing detection triptolide.
6. application as claimed in claim 1, it is characterised in that the degree of the myocardial damage will not cause myocardium LDH, cardiac muscle
CK-MB or cardiac muscle CAT activity reduction.
7. application as claimed in claim 1, it is characterised in that the degree of the myocardial damage will not cause blood plasma granzyme B
Change in concentration.
8. application as claimed in claim 1, it is characterised in that the degree of the myocardial damage will not cause blood plasma myocardium myo calcium
Albumen-I change in concentration.
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| CN201610332687.8A CN106018824B (en) | 2016-05-18 | 2016-05-18 | Purposes of the aryl hydrocarbon receptor in drug-induced heart disease biomarker is prepared |
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| CN1445366A (en) * | 2002-03-15 | 2003-10-01 | 中国医学科学院阜外心血管病医院 | New protein kinase specific to human cardiac muscle and its coded sequence |
| JP6047092B2 (en) * | 2010-07-27 | 2016-12-21 | トラスティーズ オブ ボストン ユニバーシティ | Aryl hydrocarbon receptor (AhR) modifier as a novel cancer therapy |
| CN105132565A (en) * | 2015-09-18 | 2015-12-09 | 雷桅 | Ultrasensitive molecule marker for heart disease diagnosis and classification, as well as application of ultrasensitive molecule marker |
| CN105273083B (en) * | 2015-11-17 | 2019-08-06 | 中国科学院生态环境研究中心 | Anti-human aryl hydrocarbon receptor monoclonal antibody and application thereof |
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