CN106018824A - Application of aromatic hydrocarbon receptor in preparation of drug-induced heart disease biomarker - Google Patents
Application of aromatic hydrocarbon receptor in preparation of drug-induced heart disease biomarker Download PDFInfo
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- CN106018824A CN106018824A CN201610332687.8A CN201610332687A CN106018824A CN 106018824 A CN106018824 A CN 106018824A CN 201610332687 A CN201610332687 A CN 201610332687A CN 106018824 A CN106018824 A CN 106018824A
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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 the technical field of biology, and particularly relates to an application of an aromatic hydrocarbon receptor in preparation of a drug-induced heart disease biomarker.
Description
Technical field
The invention belongs to biological technical field, particularly relate to aryl hydrocarbon receptor in preparing drug-induced heart disease biomarker
Purposes.
Background technology
In broad sense, the heart change that all myocardial toxicity direct or indirect due to medicine are caused, either because medicine is to cardiac muscle
Electrophysiological impact and the myocardium depolarization that causes and the exception of multipole or various arrhythmia, or the contractility of cardiac muscle is because of the poison of medicine
Property effect and be suppressed, and then induction or the heart failure that increases the weight of, or because certain drug allergy is caused myocarditis, pericardium
Inflammation, all can be referred to as drug-induced heart disease (drug-induced heart diseases).As other drug-induced disease, Drug
Heart disease can be facilitated due to the reason of medicine itself, the doctor of medication and the patient three controlled aspect, but generally medicine itself with
Drug interaction is the leading factor causing pathological changes.Medicine involves heart, and person is mainly the infringement to cardiac muscle, but minority medicine is also
Involve pericardium and endocardium.
Radix Tripterygii Wilfordii (Tripterygium wilfrdii Hook.f.) has another name called Caulis Fibraureae, Caulis Fibraureae wood, allusion quotation wax rattan, Gelsemium elegans Benth. etc., belongs to Wei Mao
Section bejuco.Its English name: Common Threewingnut Root, latin name: Radix Tripterygii Wilfordii
Tripterygium wilfordii HooK.f.Documents and materials record its bitter in the mouth, pungent, cool in nature, and poison, returns liver, kidney channel greatly.Have and dispel
The effects such as wind dehumidifying, promoting blood circulation to remove obstruction in the collateral, reducing swelling and alleviating pain, parasite killing removing toxic substances.The root of Radix Tripterygii Wilfordii, neck, Ye Junke are as medicinal, mainly
Medicinal part is its root.Finding in clinical practice, the mechanism of Tripterygium Preparations treatment rheumatoid arthritis is similar to corticosteroids,
But not having the side effect of hormones, better tolerance, potential applicability in clinical practice is wide.But research in recent years finds, Radix Tripterygii Wilfordii can
Causing toxic myocarditis, cause cardiac conduction obstacle, severe patient may occur in which cardiogenic shock and heart failure.At various Thunder Gods
In the acute toxic cardiac damage that vine formulations causes, the cardiac damage caused with triptolide again is the most serious.
Some researchs show, triptolide has different inhibitory action, as increase mitochondrial oxidation stress, P-glycoprotein base
The expression of cause and function, Nuclear factor kappa B (NF-κ B) signal and cyclooxygenase-2 expression, TAK1/MAP3K7 activity (is passed through
The formation of interference TAK1/MAP3K7--TAB1/MAP3K7IP1 complexity thing), lower Rac1 and JAK/STAT3 path, and
The oxidative stress of induction.Additionally, some research groups report, triptolide is transcription factor II (TFIIH) human-like RNA
The inhibitor of polymerase II.Week et al. report triptolide by mitochondria pathway increase oxidative stress and inducing cell apoptosis and
Depolarization mitochondrial membrane potential subsequently, reduces Bax/Bcl-2 ratio, release cells pigment C and activation Caspase-3.Some
Research it turned out, and in non-myocardial infarction system, Radix Tripterygii Wilfordii toxicity relates to mitochondria pathway.A nearest research shows,
Triptolide passes through transcription factor TFIIH, transcribing and Nucleotide Sequence Analysis of suppression rna plymerase ii mediation.Gene lacks
Mistake or triptolide suppression TFIIH can induce the apoptosis that the tumor cell JNK of p53 defect relies on.
Biomarker (Biomarker) refers to can be with Mk system, organ, tissue, cell and subcellular structure or function
Change or the biochemical indicator of contingent change, there is purposes widely.Biomarker can be used for medical diagnosis on disease, sentences
Disconnected staging or be used for evaluating new drug or the new therapy safety and efficacy in target group.Diagnose the heart clinically at present
The biochemical indicator of function has been widely used in the detection of cardiotoxicity of medicine.Wherein, cardiac muscle troponin I (cTnI) is one
Plant specific to myocardial cell, exist only in the albumen in ventricular muscles and heart muscle.The molecular weight of cTnI is 22ku, serum half
The phase of declining is about 2h.CTn I there are about 3%～6% in cardiac muscle and is present in a free form in endochylema, and remainder is then with knot
Conjunction form is present in sarcostyle.In normal human serum, the content of cTn I is little, falls ill in acute myocardial infarction (AMI)
After, serum cTn I starts more than 3～6h to raise, and 10～24h reach peak, and 5～7d recover normal.With cardiac muscle creatine
Kinases MB (CK-MB) compares, and cTn I content in cardiac muscle is more, and molecular weight is less than normal, after myocardial damage easily in serum
Measured, so it is the most sensitive, can be used for the microlesion of detection cardiac muscle.Additionally, have in document report cardiac muscle infiltrating cells
The expression of granzyme B becomes positive correlation with the cardiac damage order of severity.
Myocardial enzymes includes creatine kinase isozyme (CK-MB), lactic acid dehydrogenase (LDH) etc., is commonly used to joint-detection cardiac muscle
The most impaired.
Cardiac muscle CK-MB (CK-MB): detection CK-MB hypotype is for acute myocardial infarction (AMI)
Diagnosis, CK-MB has 3 kinds of hypotypes to be present in internal, and CK-MB1 is blood plasma type, CK-MB2 for tissue
Type, is present in myocardial cell, is released into blood when myocardial damage and is changed into CK-MB1.Under normal circumstances,
CK-MB2/CK-MB1 ＜ 1.0, as when ratio rises to 1.5～1.7, prompting exists myocardial damage, detects CK-MB1 and CK-MB2
Hypotype more has Sensitivity and Specificity to diagnosis AMI.Many the rising at AMI of CK-MB 3～6h raises after being ill, and 12～24h reach
Peak, 2～3d recover normal.It is one of AMI sensitive indicator in early days that CK-MB raises.
Catalase (CAT) is a kind of enzyme scavenger, is also called catalase, is the desmoenzyme with iron porphyrin as prothetic group.It
H can be promoted2O2Being decomposed into molecular oxygen and water, the hydrogen peroxide in purged body, so that cell protects against H2O2Murder by poisoning,
It it is one of the key enzyme of biophylaxis system.
Aryl hydrocarbon receptor (AHR) be one mediation xenobiotic metabolism orphan nuclear receptor, cardiovascular system growth and
Functionally there is pivotal role.The loss of AHR presents hypoxemia, and endothelin-1 expression increases, systemic hypertension, cardiac muscle
Plumpness and fibrosis.There are some researches show, internal shortage AHR causes left ventricular function to be remarkably decreased, and amycin processes and promotes heart
P53 gene activation and apoptosis increase.Except the dioxin toxic action of mediation, some researchs show that AHR also assists in human body diseases
The effect of system of defense.Some datas show, are exposed to pollutant, as PCB, B [a] P, TBT and smoke from cigarette increase
AHR expresses.But, it is but closely related with allergy that AHR expresses increase.
There are some researches show, the systemic hypertension that AHR disappearance causes and the expression increasing endothelin-1.Separately studies have reported that, lack
Weary AHR expresses the α 1D adrenoceptor promoting blood vessel and the effect strengthening Angiotensin II.Several groups of reports show,
The disappearance of AHR gene causes cardiac hypertrophy.On the contrary, glucose is taken in and can be dramatically increased heart AHR and Nrf2 activity, improves
The heart failure that spontaneous hypertensive rat is adjoint.
Summary of the invention
The purpose of the present invention aims to provide AHR purposes in preparing drug-induced heart disease biomarker.
Specifically, a first aspect of the present invention there is provided aryl hydrocarbon receptor at preparation detection Radix Tripterygii Wilfordii or Radix Tripterygii Wilfordii extract
Application in the biomarker of the myocardial damage caused, and described Radix Tripterygii Wilfordii or Radix Tripterygii Wilfordii extract with triptolide be
Active component.
In a preference, the degree of described myocardial damage will not cause cardiac muscle LDH, cardiac muscle CK-MB or cardiac muscle CAT
Activity reduction.
In another preference, the degree of described myocardial damage will not cause the concentration change of blood plasma granzyme B.
In another preference, the degree of described myocardial damage will not cause the concentration change of blood plasma Cardiac Troponin-I.
A second aspect of the present invention there is provided the life of the myocardial damage that aryl hydrocarbon receptor causes at preparation detection triptolide
Application in thing mark.
In a preference, the degree of described myocardial damage will not cause cardiac muscle LDH, cardiac muscle CK-MB or cardiac muscle CAT
Activity reduction.
In another preference, the degree of described myocardial damage will not cause the concentration change of blood plasma granzyme B.
In another preference, the degree of described myocardial damage will not cause the concentration change of blood plasma Cardiac Troponin-I.
The details of various aspects of the present invention will be able to detailed description in chapters and sections subsequently.By hereafter and the description of claim,
The feature of the present invention, purpose and advantage will become apparent from.
Accompanying drawing explanation
Fig. 1 is once to rat triptolide 1mg/kg, the metamorphosis (HE dyeing) of rat heart muscle tissue after 14 days
Fig. 2 is once to rat triptolide 1mg/kg, uses LDH and CK-BM and detect with CAT test kit after 14 days
The activity of LDH, CK-BM and CAT in heart
Fig. 3 is once to rat triptolide 1mg/kg, respectively with Cardiac Troponin-I test kit and rat granule after 14 days
Enzyme B (GzmB) kit measurement Cardiac Troponin-I and the concentration of rat granzyme B
Fig. 4 is once to rat triptolide 1mg/kg, with in rat aryl hydrocarbon receptor kit measurement blood plasma and cardiac muscle after 14 days
The concentration of AHR
Fig. 5 is once to rat triptolide 1mg/kg, CYP1A1 during real-time quantitative RT-PCR measures rat heart muscle after 14 days
The expression of (a kind of p450 enzyme by AHR regulating and expressing)
Fig. 6 is that after 14 days, the form of rat liver (A) and kidney (B) tissue becomes once to rat triptolide 1mg/kg
Change (HE dyeing)
Fig. 7 is once to rat triptolide 1mg/kg, with LDH and CK-BM test kit detection liver (A, C) after 14 days
Activity with kidney (B, D) interior LDH and CK-BM
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments be merely to illustrate the present invention and not
For limiting the scope of the present invention.Reagent and raw material used in the following example all can be bought by commercial sources and obtain.Under
The experimental technique of unreceipted actual conditions in row embodiment, generally according to normal condition or according to the condition proposed by manufacturer.
Unless otherwise defined, the same meaning that all specialties used in literary composition are familiar with one skilled in the art with scientific words.This
Outward, any method similar or impartial to described content and material all can be applicable in the inventive method.Preferable described in literary composition
Implementation only presents a demonstration with material and is used.
The features described above that the present invention mentions, or the feature that embodiment is mentioned can be in any combination.Institute disclosed in patent specification
There is the feature can be with any composition forms use, each feature disclosed in description, can any provide identical, impartial
Or the alternative characteristics of similar purpose replaces.Therefore except there being special instruction, disclosed feature is only equalization or the one of similar features
As property example.
Embodiment 1 triptolide toxic action to rat heart
1.1 purposes: observe once heavy dose of Cardiotoxicity to triptolide.
1.2 experiment materials:
1.2.1 reagent: CMC-Na is purchased from Shanghai section good medicine inspection equipment purchased from Chemical Reagent Co., Ltd., Sinopharm Group, triptolide
Company limited.
1.2.2 laboratory animal and feedstuff: SD rat, male, body weight (120g～140g), of the right age, healthy.Western general purchased from Shanghai
Er-Bi Kai laboratory animal company limited, animal credit number: SYXK (Shanghai) 2013-0058.Animal feed irradiation Mus material is originated
In Shi Lin bio tech ltd, Shanghai, Feed Manufacturing licence: Shanghai raises careful (2008) 04031.
1.2.3 experimental apparatus: superspeed refrigerated centrifuge Centrifuge 5810R is purchased from Eppendorf company of Germany
1.3 experimental techniques:
SD rat is randomly divided into matched group and experimental group (n=8), disposably gives triptolide 1mg/kg (0.5%
CMC-Na is solvent).Observe the mortality rate of animal, signs of toxicity, food consumption, chaeta outward appearance and the change of behavior every day.
Dissect after 14 days, take blood and isolating cardiac, liver, kidney and other organs.Use the sodium citrate (1:9 use) of 3.8%
As blood anticoagulant.Blood is centrifuged 10 minutes under 4 DEG C and 1000 × g, it is thus achieved that after blood plasma, subpackage is stored in-80 DEG C of ice
In case stand-by.The heart, liver, nephridial tissue sections stained with hematoxylin and Yihong (H&E) dyeing.
1.4 experimental results:
Tissue slice result shows, compared with the control, triptolide cause Cardiomyocytes Hypertrophy, edema and be partly dissolved,
Intercellular substance increases, cardiac muscle fiber fracture, wherein male rat even more serious than the myocardial damage of female rats (Fig. 1), and
Under identical test dosage, no abnormal change is at liver and kidney (Fig. 6).
1.5 conclusions: once heavy dose can cause myocardiac histology pathological changes to triptolide.There is sex difference in this toxicity.
Enzyme activities in the cardiac muscle that embodiment 2 myocardial damage causes
2.1 purposes: once heavy dose of give triptolide in the case of, observe myocardial damage and cause the change of enzymatic activity in cardiac muscle.
2.2 experiment materials:
2.2.1 reagent: rat creatine kinase isozyme (CK-MB) ELISA kit, rat lactic acid dehydrogenase (LDH), rat
Catalase (CAT) detection kit is all built up Bioengineering Research Institute and is bought from Nanjing
2.2.2 laboratory animal and feedstuff: with embodiment 1.2
2.2.3 experimental apparatus: superspeed refrigerated centrifuge Centrifuge 5810R is purchased from Eppendorf company of Germany；Microplate reader light is inhaled
Receive the microplate reader SpectraMax 190 Molecular Devices company purchased from the U.S.；The full-automatic quick beveller of sample is purchased
From Shanghai Jing Xin Industrial Co., Ltd.；Ultrasonic cell disruptor is purchased from NingBo XinZhi Biology Science Co., Ltd
2.3 experimental technique
2.3.1 zoopery: with embodiment 1.3
2.3.2 tissue homogenate preparation and enzyme assay
Core, liver and nephridial tissue specimen, with the tissue weight (g) of 1:9 ratio: normal saline (ml) in beveller (at liquid nitrogen
Under cooling) grind, setpoint frequency 75HZ, continue 50 seconds, grind 2 times, take out tissue fluid.The most ultrasonic 5 times, each 2
Second.Then, it is centrifuged at 4 DEG C 10 minutes, 2500 revs/min, takes supernatant, dilution and subpackage, cardiac muscular tissue's liquid of 10% is put in
-80 degree Refrigerator stores.Tissue fluid protein concentration, rat creatine kinase isozyme (CK-MB) ELISA is surveyed with Coomassie Brilliant Blue
Test kit, rat lactic acid dehydrogenase (LDH), the enzyme assay foundation kit manufacturer of rat catalase (CAT)
Designation method is carried out.
2.3.3 data process and statistical analysis
Using GraphPad Prism 5 software or SPSS15.0 software to carry out data process, data are with mean ± standard deviation
(mean ± SD) represents, compares employing pairing t-inspection and carries out statistical analysis, compare employing single factor test variance and divide in group between group
Analysis method (one-way ANOVA) analyzes and processes, and p 0.05 thinks there is significant difference, has statistical significance.
2.4 experimental results:
To under triptolide 1mg/kg test dose, compared with matched group, myocardium of male rat LDH, CK-MB, CAT
Activity substantially reduces, and there are no significant the change of the index of female rats, this result is consistent with the HE coloration result of 1.4, explanation
Female rats myocardial damage is not serious (Fig. 2).Additionally, the change of the liver of male and female rat or kidney LDH and CK-MB is failed to understand
Aobvious, this result is consistent with the HE coloration result of 1.4, illustrates that the liver of male and female rat or kidney the most substantially damage (Fig. 7).
2.5 conclusions: once heavy dose of give triptolide, only when cardiac muscle is by major injury, damage markers LDH, CK-MB,
CAT activity just can show significant change.There is sex difference in the testing result of these toxicity indexs.
Blood plasma Cardiac Troponin-I (cTnI) that embodiment 3 myocardial damage causes and rat granzyme B (GzmB) concentration change
3.1 purposes: in the case of once heavy dose gives triptolide, observe myocardial damage biomarker Cardiac Troponin-I
Change with rat granzyme B (GzmB).
3.2 experiment materials:
3.2.1 reagent: rat heart muscle troponin-i enzyme-linked immunosorbent assay kit is purchased from Lifeassays AB of the U.S. (Life
Diagnostics,Inc.).Rat granzyme B (GzmB) ELISA kit is purchased from the Wuhan limited public affairs of Yi Lairuite biotechnology
Department
3.2.2 laboratory animal and feedstuff: with embodiment 1.2
3.2.3 experimental apparatus: superspeed refrigerated centrifuge Centrifuge 5810R is purchased from Eppendorf company of Germany；Microplate reader light is inhaled
Receive the microplate reader SpectraMax 190 Molecular Devices company purchased from the U.S.
3.3 experimental technique
3.3.1 zoopery: with embodiment 1.3
3.3.2 blood plasma Cardiac Troponin-I concentration measures
The operating procedure that the test of blood plasma Myocardial troponin-i is specified according to production company, in 96 orifice plates, is separately added into
Standard substance or sample diluting liquid 200 μ L, cover film, hatch 2 hours for 25 DEG C.Discard liquid in hole, wash 5 times, inhale
Dry every hole residual liquid, after adding anti-cTnl antibody diluent 100 μ L, addition HRP enzyme enzyme conjugates working solution 100ml, 25 DEG C
Hatch 1 hour.Washing 5 times, blot, every hole adds zymolyte TMB100 μ L, after overlay film, hatches 20 minutes for 25 DEG C
After, add stop buffer 100 μ L, terminate reaction.Immediately by the microplate reader OD value in each hole of 450nm wavelength measurement.
3.3.3 blood plasma rat granzyme B (GzmB) concentration measures
The maneuvering sequence specified according to production company, in 96 orifice plates, is separately added into standard substance or sample diluting liquid 100 μ L, respectively
Hole is separately added into standard substance or testing sample 100 μ L, cover film, hatches 90 minutes for 37 DEG C.Discard liquid, add biotin
Change antibody working solution 100 μ L, 37 DEG C of incubations 1 hour.Discard liquid in hole, wash 3 times, blot residual liquid.Add enzyme to combine
Thing working solution 100 μ L, 37 DEG C of incubations 30 minutes.Every hole adds zymolyte TMB solution 90 μ L, and at 37 DEG C, lucifuge is hatched
After about 15 minutes, add stop buffer 50 μ L, terminate reaction.By 450nm wavelength measurement OD value.
3.3.4 data process and statistical analysis
Using GraphPad Prism 5 software or SPSS15.0 software to carry out data process, data are with mean ± standard deviation
(mean ± SD) represents, compares employing pairing t-inspection and carries out statistical analysis, compare employing single factor test variance and divide in group between group
Analysis method (one-way ANOVA) analyzes and processes, and p 0.05 thinks there is significant difference, has statistical significance.
3.4 experimental results:
To under triptolide 1mg/kg test dose, detect the concentration of blood plasma cTnI and GzmB.Compared with matched group,
Rat GzmB there are no significant change (Fig. 3 B).Only male rat blood plasma cTnI concentration shows and dramatically increases, and female
In property rat plasma, cTnI there was no significant difference (Fig. 3 A).
3.5 conclusions: once heavy dose of give triptolide after, quick to myocardial damage of the concentration of blood plasma rat granzyme B (GzmB)
Sensitivity is inconspicuous, and blood plasma Cardiac Troponin-I level exists sex difference.
AHR level change in blood plasma that embodiment 4 myocardial damage causes and cardiac muscle
4.1 purposes: once heavy dose of give triptolide in the case of, observe AHR egg in the blood plasma and cardiac muscle that myocardial damage causes
White level changes.
4.2 experiment materials:
4.2.1 reagent: rat aryl hydrocarbon receptor ELISA kit is purchased from Beijing equation one hundred gold medal Science and Technology Ltd.
4.2.2 laboratory animal and feedstuff: with embodiment 1.2
4.2.3 experimental apparatus: superspeed refrigerated centrifuge Centrifuge 5810R is purchased from Eppendorf company of Germany；Microplate reader light is inhaled
Receive the microplate reader SpectraMax 190 Molecular Devices company purchased from the U.S.；The full-automatic quick beveller of sample is purchased
From Shanghai Jing Xin Industrial Co., Ltd.；Ultrasonic cell disruptor is purchased from NingBo XinZhi Biology Science Co., Ltd
4.3 experimental technique
4.3.1 zoopery: with embodiment 1.3
4.3.2 prepared by tissue homogenate
Core dirty tissue specimen, with the tissue weight (g) of 1:9 ratio: PBS (PH7.4) (ml) in beveller (cold at liquid nitrogen
But under) grind, setpoint frequency 75HZ, continue 50 seconds, grind 2 times, take out tissue fluid.The most ultrasonic 5 times, each 2 seconds.
Then, it is centrifuged 20 minutes at 4 DEG C, 2500 revs/min, takes supernatant, subpackage, cardiac muscular tissue's liquid of 10% is put in-80 degree refrigerators
Preserve.Tissue fluid protein concentration is surveyed with Coomassie Brilliant Blue.
4.3.3AHR concentration measures
The operating procedure specified according to production company, set respectively blank well (blank control wells is 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 30 of shrouding film shrouding
Minute.By standby after 20 times of concentrated cleaning solution distilled water 20 times dilution.Discarding liquid, dry, cleaning mixture is filled it up with in every hole,
Discard after standing 30 seconds, be so repeated 5 times, pat dry.Every hole adds enzyme marking reagent 50 μ l, except blank well, 37 DEG C of incubations
30 minutes.Discard liquid, dry, wash 5 times.Every hole is initially charged developer A50 μ l, adds developer B50 μ l, gently
Gently shaking mixing, 37 DEG C of lucifuges develop the color 10 minutes.Every hole adds stop buffer 50 μ l, terminates reaction (now blue standing turns yellow).
Returning to zero with blank well, 450nm wavelength sequentially measures the OD value in each hole.
4.3.3 data process and statistical analysis
Using GraphPad Prism 5 software or SPSS15.0 software to carry out data process, data are with mean ± standard deviation
(mean ± SD) represents, compares employing pairing t-inspection and carries out statistical analysis, compare employing single factor test variance and divide in group between group
Analysis method (one-way ANOVA) analyzes and processes, and p 0.05 thinks there is significant difference, has statistical significance.
4.4 experimental results:
Fig. 4 is under triptolide 1mg/kg test dose, the concentration of AHR in detection blood plasma and cardiac muscle.With matched group
Comparing, in rat heart and blood, AHR protein level has significance to decline.And 2.4 results show that Liver and kidney is without substantially damage, institute
It is not derived from Liver and kidney with AHR, illustrates that the cardiac toxicity that triptolide causes can make AHR albumen water in heart and blood
Flat have significance to decline.
4.5 conclusions: aryl hydrocarbon receptor AHR can be as the detection cardiopathic biomarker of Radix Tripterygii Wilfordii property.
In embodiment 5 applies real-time quantitative RT-PCR detection cardiac muscle, CYP1A1 expresses change.
5.1 purposes: the expression of the downstream gene CYP1A1 of AHR in detection cardiac muscle, to judge that can AHR as triptolide
The early monitoring of cardiac toxicity and evaluation index
5.2 experiment materials:
5.2.1 laboratory animal and feedstuff: with embodiment 1.2
5.2.2 reagent: Maxima SYBR Green qPCR Master Mix, Invitrogen Trizol reagent and High-Capacity
CDNA Reverse Transcription Kit is all purchased from purchased from U.S.'s life technology (Life Technologies) company；Gene specific
Property primer by Shanghai JaRa bio-engineering corporation synthesize.
5.2.3 experimental apparatus: the full-automatic quick beveller of sample is purchased from Shanghai Jing Xin Industrial Co., Ltd.；Ultrasonic cell disruptor is purchased from
NingBo XinZhi Biology Science Co., Ltd；Superspeed refrigerated centrifuge Centrifuge 5810R is purchased from Eppendorf company of Germany；
PCR reverse transcription instrument (thermocycler, Biometra company of Germany)；ABI 7500fast PCR amplification instrument.
5.3 experimental techniques:
5.3.1 zoopery: with embodiment 1.3
5.3.2 real-time quantitative PCR
5.3.2.1 Total RNAs extraction
5.3.2.1.1 the step extracting cardiac muscular tissue's total serum IgE is:
1) weigh cardiac muscular tissue 30-40mg to be put in the EP pipe of 2ml, add 2 steel balls, add the Trizol reagent of 1ml,
It is put in tissue grinder instrument, beveller adds liquid nitrogen, grinds, take out steel ball.Ultrasound for homogenization, is spaced the ultrasonic 4s of 2s,
Each sample surpasses 4-5 time, and room temperature stands 5min.Centrifugal (4 DEG C, 12000rpm, 10min), takes supernatant.
2) often pipe adds 0.2mL chloroform, and fully vibrate 15s, and room temperature stands 15min；
3) be divided into three layers after centrifugal (4 DEG C, 12000rpm, 15min), transfer about 450 μ L upper strata aqueous phases to new 1.5mL from
In heart pipe, and adding equal-volume isopropanol, for several times, room temperature places 15min in reverse mixing；
4) centrifugal (4 DEG C, 12000rpm, 10min), abandon supernatant, it is seen that white precipitate at the bottom of pipe；
5) add 1mL75% washing with alcohol once, 4 DEG C, 12000rpm be centrifuged 5min, abandon supernatant；
6) add 1mL absolute ethanol washing once, after being centrifuged, abandon supernatant, in super-clean bench, be dried 10min；
7) the water dissolution precipitation that about 15-25 μ L DEPC processed is added；
8) after 55 DEG C of water-bath 5min, rifle head blows and beats mixing sample repeatedly, therefrom draws 1 μ L and is diluted to 100 μ L, ultraviolet determination
The concentration of RNA.
5.3.2.2 reverse transcription synthesis cDNA
Illustrate according to Reverse Transcription box, the total serum IgE of extraction is carried out reverse transcription.
Each reagent is after thawing on ice, and on ice, the reverse transcription system as shown in table 1 operates:
Table 1 reverse transcription system
After each reactant liquor and sample are mixed, it is centrifuged and removes bubble.Putting samples in PCR instrument, reverse transcription reaction program is set to 25 DEG C
× 10min, 37 DEG C × 2h, 85 DEG C × 5min, 16 DEG C × 24h.The cDNA that reverse transcription obtains is placed in-80 DEG C of preservations.
5.3.2.3PCR amplification
According to description, PCR reaction system is as shown in table 2:
Table 2 PCR reaction system
Putting in PCR instrument after sample blending is centrifugal, response procedures is: 95 DEG C × 30s carries out denaturation, then two-step method (95 DEG C
× 3s → 60 DEG C × 30s, 40 circulations) expand.The Ct value of the Ct GAPDH of genes of interest is standardized by result,
Calculate gene expression relative quantity according to relative method, use formula 2-Δ Δ Ct to calculate relative fold's change of gene expression.
Primer sequence see table:
Table 3 quantitative PCR measures primer
5.3.3 data process and statistical analysis
Using GraphPad Prism 5 software or SPSS15.0 software to carry out data process, data are with mean ± standard deviation
(mean ± SD) represents, compares employing pairing t-inspection and carries out statistical analysis, compare employing single factor test variance and divide in group between group
Analysis method (one-way ANOVA) analyzes and processes, and p 0.05 thinks there is significant difference, has statistical significance.
5.4 experimental results:
Fig. 5 is under triptolide 1mg/kg test dose, detects under the interior AHR of cardiac muscle with real-time quantitative RT-PCR
The mrna expression of trip Gene CYP1A1, data represent the mean+SD of at least three independent experiment.
5.5 brief summaries:
To under triptolide 1mg/kg test dose, by the mrna expression of the p450 enzyme subtype C YP1A1 that AHR regulates and controls
Being all substantially to reduce in male and female rat heart, this is consistent with the conclusion in 4.5.So, AHR can be the Radix Tripterygii Wilfordii property heart
Disease of ZANG-organs diagnosis provides and selects.
Many aspects involved in the present invention have been done as explained above.However, it should be understood that without departing from present invention spirit
Putting, those skilled in the art can carry out equivalent to it and change and modify, and described change and modification fall into this Shen equally before
Please the coverage of claims.
Claims (8)
1. aryl hydrocarbon receptor is in the biomarker of myocardial damage that preparation detection Radix Tripterygii Wilfordii or Radix Tripterygii Wilfordii extract cause
Application, and described Radix Tripterygii Wilfordii or Radix Tripterygii Wilfordii extract are with triptolide as active component.
Apply the most as claimed in claim 1, it is characterised in that the degree of described myocardial damage will not cause cardiac muscle
The activity reduction of LDH, cardiac muscle CK-MB or cardiac muscle CAT.
Apply the most as claimed in claim 1, it is characterised in that the degree of described myocardial damage will not cause blood plasma
The concentration change of Cytotoxic cell proteinase-1.
Apply the most as claimed in claim 1, it is characterised in that the degree of described myocardial damage will not cause the blood plasma heart
The concentration change of flesh troponin-i.
5. aryl hydrocarbon receptor application in preparation detects the biomarker of the myocardial damage that triptolide causes.
Apply the most as claimed in claim 1, it is characterised in that the degree of described myocardial damage will not cause cardiac muscle
The activity reduction of LDH, cardiac muscle CK-MB or cardiac muscle CAT.
Apply the most as claimed in claim 1, it is characterised in that the degree of described myocardial damage will not cause blood plasma
The concentration change of Cytotoxic cell proteinase-1.
Apply the most as claimed in claim 1, it is characterised in that the degree of described myocardial damage will not cause the blood plasma heart
The concentration change of flesh troponin-i.
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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 |
| WO2012015914A2 (en) * | 2010-07-27 | 2012-02-02 | Trustees Of Boston University | Aryl hydrocarbon receptor (ahr) modifiers as novel cancer therapeutics |
| 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 |
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| WO2012015914A2 (en) * | 2010-07-27 | 2012-02-02 | Trustees Of Boston University | Aryl hydrocarbon receptor (ahr) modifiers as novel cancer therapeutics |
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