CN109797129A - Nrf2/ARE expressed in hypoxemia and with astragalus polyose protective effect measuring method - Google Patents
Nrf2/ARE expressed in hypoxemia and with astragalus polyose protective effect measuring method Download PDFInfo
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Abstract
The invention belongs to Nrf2/ARE signal path regulating and controlling effect detection technique field, disclose a kind of Nrf2/ARE expressed in hypoxemia and with astragalus polyose protective effect measuring method；Using adherent method culture HPAECs, establish the anoxia model of HPAECs, between measuring different groups in HPAECs supernatant Nrf2, SOD, GSH concentration；The expression of RT-PCR detection endothelial cell Nrf2mRNA, SODmRNA, GSH-PXmRNA；Western blot determination Nrf2, SOD, GSH-PX protein content.The expression that the present invention passes through measurement body itself anti-oxidation stress signal path Nrf2/ARE HPAECs in hypoxemia, speculate the high expression in pulmonary artery endothelial cell of Nrf2 albumen, and astragalus polyose can play anti-oxidative stress by the high expression of enhancing Nrf2 albumen, and function and effect have time-and concentration-dependent.
Description
Technical field
The invention belongs to Nrf2/ARE signal path regulating and controlling effect detection technique fields more particularly to a kind of Nrf2/ARE to exist
When hypoxemia expression and with astragalus polyose protective effect measuring method.
Background technique
Currently, the prior art commonly used in the trade is such that COPD is a kind of common disease, more for seriously endangering human health
Morbidity, case fatality rate is high, brings heavy financial burden to patient, society, China adjusts 7 20245, area adults
It looks into, the illness rate of COPD is up to 8.2% in 40 years old or more crowd as the result is shown, the World Bank and World Health Organization's data sheet
Bright, to 2020, COPD will occupy world's disease financial burden the 5th.The core of Keapl/Nrf2-ARE signal transduction pathway
Molecule includes NF-E2 correlation factor -1 (NF-E2-related factor 2, Nrf2), Kelch sample ECH associated protein 1
(Keap1) and Antioxidant responsive element (antioxident response element, ARE), in cytoplasm Nrf2 with
Keap1 is combined, and is anchored on the cytoskeleton being made of actin by Keap1 albumen, when the thorn by ROS etc.
When swashing, Keap1 and Nrf2 uncoupling make Nrf2 be transferred to core, with the Maf protein binding in gene at identifying after heterodimer
ARE.Both at home and abroad multiple studies have shown that Keapl/Nrf2-ARE signal transduction pathway takes part in nervous system, endocrine system, swells
The reaction of itself anti-oxidation stress of tumor system generates II phase detoxication enzyme by induction body and the high of anti-oxidant albumen is expressed, antagonism
The oxidative damage of tissue.Sulforaphane can activate Nrf2 signal path, raise antioxidant enzyme and quinone redox downstream
The expression of the enzymes such as enzyme 1 (NQO-1) mitigates blood-CSF barrier oxidative damage.Low dosage oxidant active cell Nrf2, can be with
The induced oxidative stress effect of the chemical reagent such as arsenic and hypochlorous acid exposure is resisted, oxidativestress damage is protected cells from.It is high
Blood glucose induces Nrf2- depleted mice response to oxidative stress, leads to renal dysfunction, above-mentioned change can be reversed after activating Nrf2.
And Nrf2 knockout rat, diabetic cardiomyopathy can also be led in very short time by only slightly increasing even if blood glucose, equally
By activating Keapl/Nrf2-ARE access, diabetic cardiomyopathy process can be prevented.It is now recognized that Nrf2 activation is that cell is resisted
The committed step of oxidative stress, but there is no research shows that participate in the response to oxidative stress of COPD, COPD merge pulmonary hypertension,
In the pathophysiological change of pulmonary heart disease, the conditions associated with hypoxia damage of HPAECs plays a significant role, and by Keap1/Nrf2/ARE
The regulation of access is likely to become the important method for preventing and treating COPD complication.
In conclusion problem of the existing technology is:
(1) adherent method culture HPAECs is used for the first time, the anoxia model of HPAECs is established, between measuring different groups on HPAECs
The concentration of Nrf2, SOD, GSH-PX in clear liquid；
(2) for the first time using the expression of RT-PCR detection endothelial cell Nrf2mRNA, SODmRNA, GSH-PXmRNA；
(3) western blot determination Nrf2, SOD, GSH-PX protein content.
Solve the difficulty and meaning of above-mentioned technical problem: the present invention passes through measurement body itself anti-oxidation stress signal path
The expression of Nrf2/ARE HPAECs in hypoxemia, thus it is speculated that the high expression in pulmonary artery endothelial cell of Nrf2 albumen, and also Radix Astragali is more
Sugar can play anti-oxidative stress by the high expression of enhancing Nrf2 albumen, and function and effect have time and concentration dependant
Property, effectively prolong the course of disease that COPD merges pulmonary hypertension, pulmonary heart disease.And then by Nrf2/ARE signal path in hypoxemia
The research of HPAECs expression inquires into it in COPD and the mechanism of action in oxidative stress occurs, high to delay COPD that pulmonary artery occurs
Pressure, pulmonary heart disease provide theoretical foundation.This area old age COPD disease incidence rises year by year, and patient and social economical burden are increasingly heavy
Weight, and astragalus polyose system traditional medicine, cheap, toxic side effect is few, if national medicine strong point can be played, promotes it at this
The therapeutic effect in field actively provides treatment new approaches for this area COPD merging pulmonary hypertension, pulmonary heart disease, to play this
Regional Chinese medicine therapeutic features make patient be benefited, strive for maximum social benefit.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of Nrf2/ARE expresses and more with Radix Astragali in hypoxemia
Sugared protective effect measuring method.
The invention is realized in this way a kind of Nrf2/ARE expressed in hypoxemia and with astragalus polyose protective effect measurement side
Method, the Nrf2/ARE are expressed in hypoxemia and are included: with astragalus polyose protective effect measuring method
The first step establishes the anoxia model of HPAECs using adherent method culture HPAECs, between measuring different groups on HPAECs
The concentration of Nrf2, SOD, GSH-PX in clear liquid；
Second step, RT-PCR detect endothelial cell Nrf2mRNA, SOD mRNA, GSH-PXmRNA, expression；
Third step, western blot determination Nrf2, SOD, GSH-PX protein content.
Further, the first step specifically includes:
2) recovery of HPAECs cell is frozen
1. being immediately placed in 37 DEG C of water-baths, and gently shake cell cryopreservation tube is taken out in liquid nitrogen container, melting it quickly
Change；
2. 1000rpm is centrifuged 5min, is opened after disinfection, suck supernatant with pipette tips；
3. 1640 culture mediums of the cell precipitation with lml containing 10% newborn bovine serum are resuspended, cell suspension moves into 25ml culture
Bottle adds culture solution to total volume to 5ml；
4. being placed in 37 DEG C, 5%CO2, cultivated in saturated humidity cell incubator, for 24 hours after seen under inverted microscope
Examine cell whether adherent growth, replace culture solution；
2) cell passes on
1. 2-3 days replacement culture solutions of the cell of adherent growth are primary, cell, which converges when piece reaches 70-80%, to be passed on；
2. aspirator sucks the liquid in culture dish, 0.25% trypsin solution of 37 DEG C of 1ml preheatings is added,
Microscopically observation is set to after shrinking attached cell, space between cells becomes larger, cell is become round by adherent polygon, removal
Trypsase；
3. being rapidly added 1640/DMEM culture medium, trypsin digestion effect is terminated.Being blown and beaten repeatedly with 3ml liquid-transfering gun makes
Attached cell suspends；
4. cell suspension equivalent is moved into 2-3 25ml culture bottle, it is added into each culture bottle and contains 10% new born bovine
1640 culture medium of serum is placed in 37 DEG C, 5%CO to total amount about 5ml2Saturated humidity cell incubator in continue to cultivate.
The culture solution of HPAECs cell uses 1640 culture mediums, dual anti-, penicillin concn is added are as follows: 100U/ml, streptomysin concentration are
100μg/ml；
3) freezing for cell is cultivated:
1. logarithmic growth phase cell, discards culture solution；
2. being added after 0.25% trypsin digestion and containing 10% newborn bovine serum culture medium, blow and beat repeatedly adherent thin
Born of the same parents；
3. cell suspension is moved into the sterile centrifugation tube of 15ml point bottom, 1000rpm is centrifuged 10min, and freezing of preparing is added
Culture solution contains 10%DMSO, makes the ultimate density 5x10 of cell in frozen stock solution6/ml；
4. being moved into cryopreservation tube with suction pipe, it is put into program cooling freezing storing box, moves into liquid nitrogen container and saves；
4) establish cobalt chloride HPAECs hypoxia model, HPAECs is randomly divided into five groups: blank control group, hypoxia inducible group,
Hypoxemia+basic, normal, high the intervention group of various concentration astragalus polyose；
1. blank control group: culture plate being placed in room temperature incubator, is added into culture plate and contains 10% blank drug blood
Clear DMEM/F12 culture solution；
2. hypoxia inducible group: culture plate being placed in room temperature incubator, with cobalt chloride effect l2 hours of 0.1%, simultaneously
The DMEM/F12 culture solution for containing 10% blank drug serum is added into culture plate；
3. the low middle and high concentration group of astragalus polyose Contained Serum: small with 0.1% cobalt chloride effect l2 in room temperature incubator
When, the DMEM/F12 containing 1mmol/L, 5mmol/L, 10mmol/L astragalus polyose drug serum is added into culture plate simultaneously respectively
Culture solution；Using adherent method culture HPAECs: HPAECs cell strain being recovered, every other day replacement culture solution is primary, to bottom of bottle
Covering about 80% selects well-grown 3-5 generation cell to be tested with trypsin digestion secondary culture；
5) Nrf2, SOD, GSH-PX in HPAECs supernatant between ELISA measures different groups；
1. is coated with buffer solution cell conditioned medium with the carbonate of 50mM, makes tested factor concentration 10-20 μ g/ml, add
100 holes μ l/ to 96 hole elisa Plates, 4 DEG C stand overnight；
2. after second day discards coating buffer, being washed 3 times with PBST, it is small that 37 DEG C of 150 μ l 1%BSA closings 1 are added in every hole
When；
After 3. .PBST is washed 3 times, the supernatant of 100 μ l difference doubling dilution degree is added in every hole, and control sample is added, and 37
DEG C be incubated for 2 hours；
4. after .PBST is washed 5 times, the secondary antibody of the HRP label after 100 μ l dilution is added, 37 DEG C are incubated for 1 hour；
5. after .PBST is washed 5 times, after chromogenic reagent 20min, A405 absorption value is read in microplate reader.
Further, the second step specifically includes:
1) cell total rna extracts
1. sample treatment: after exhausting culture solution, rinsing the cell of adhere-wall culture twice with sterile saline；Cracking: every
Kind sample adds 1ml Trizol reagent to be cracked；
2. layering: 15~30 DEG C are placed 5 minutes, and nucleoprotein complex is promoted to will be completely dissociated, and 0.2ml is added in 1ml Trizol
Chloroform acutely shakes 15 seconds, and 15~30 DEG C are placed 2~3 minutes, and centrifugal rotational speed is no more than 12,000g and is centrifuged 15 minutes, by supernatant
Liquid is transferred in new centrifuge tube；
3. RNA precipitate: 0.5ml isopropanol is added by 1ml Trizol reagent in supernatant and mixes, 15~30 DEG C of placements
10 minutes, at 2~8 DEG C, centrifugal rotational speed was no more than 12,000g and is centrifuged 10 minutes abandoning supernatants；
4. RNA is washed: 75% ethyl alcohol of 1ml is at least added in 75% ethanol washing RNA precipitate, 1ml Trizol reagent, saves
It is spare in -20 DEG C；
5. RNA dissolve: oscillation mix, centrifugal rotational speed be no more than 7,500g be centrifuged 5 minutes 2~8 DEG C；Room temperature or vacuum drying
RNA precipitate is redissolved in DEPC water；
6. RNA concentration and purity testing: detection RNA concentration and purity；
2) reverse transcription step
1. making ice, operates operate on ice if not otherwise indicated below；
2. short term oscillation, centrifugation mix after RNA sample and related reagent thaw；
3. being detected for mRNA level in-site, related reagent is incorporated in thin-walled without in enzyme PCR pipe according to table 1, carries out reverse transcription
Experiment:
4. 37 DEG C of 15min of reverse transcription；85℃ 5sec；
3)Real-time PCR
1. GAPDH primer sequence is as follows；
Nrf2:acacggtccacagctcatc (F)；tgtcaatcaaatccatgtcctg(R)；
GSH-PX:accggagaccaggtgatgag(F)；cgatgtcaggctcgatgtca(R)；
SOD:aaagatggtgtggccgatgt(F)；caagccaaacgacttccagc(R)；
GAPDH:ccacccatggcaaattccatggca(F)；tctacacggcaggtcaggtccacc(R)；
2. thin-walled is added without in enzyme PCR pipe in related reagent, real-time PCR reaction is carried out；
3. the setting of PCR amplification condition.
Further, the third step specifically includes:
Western-blotting detection:
1) cell prepares:
1. endothelial cell is with 5 × 105The density bed board of cells/well after adherent, replaces 1640 culture medium of serum-free；
2. after .24 hours, replacing complete medium, cell is stimulated with various concentration astragalus polyose；
3. after .30 minutes, discarding supernatant liquid, ice PBS is washed three times；
4. extracts total protein with RIPA liquid lytic cell；
5. .BCA method, which surveys 00, determines total protein concentration, sample is added and is mixed with 5 × albumen sample-loading buffer with 4:1 ratio and boils
10 minutes spare；
2) match glue: preparing 10% separation gel；After mixing, separation gel is injected in ready two glass plates crack, and small
The covering of 1ml deionized water is added in the heart in glue surface；After gel natural coagulation, deionized water is poured out in inclination, and is blotted with filter paper；
Prepare 5% concentration glue；And it is horizontally inserted into comb in two glass plate cracks, concentration glue is injected into separation gel upper layer, not stay gas
Bubble；
3) SDS-PAGE electrophoresis: using 70V voltage when bromophenol blue moves to separation gel after change 120V voltage to bromophenol blue move
It when to from bottom, cuts off the power, stops electrophoresis；
4) transferring film: electrophoresis terminates, and glue is peeled, and cuts spacer gel, carries out front and back sides in the upper right corner and marks, buffers in transferring film
15min is impregnated in liquid；It measures and cuts pvdf membrane one of the same size and open, also in prime marks and Whatman 3MM filter paper
6；Successively put sponge, 3 Whatman filter paper, pvdf membrane, SDS-PAGE glue, 3 Whatman filter paper well from anode to cathode
And sponge, it is inserted into transferring film slot, 310mA constant current, 100min；Transferring film finishes, and removes pvdf membrane, the dyeing of 1 × Ponceaux dyeing liquor
Transferring film effect is observed, PBST washes away dye liquor；
5) it closes: the pvdf membrane shifted is placed in 0.5%BSA confining liquid, room temperature is slow to shake 2h；
6) primary antibody is incubated for: after closing, film is put into PBST, slow to shake cleaning 10min × 3 time；Add primary antibody in reserving it
In 0.5%BSA confining liquid, after piping and druming mixes, drop in wet box on PARAFILM film, pvdf membrane being faced down and is laid on thereon, and 4
DEG C overnight；
7) secondary antibody is incubated for: after primary antibody is incubated for, film is put into PBST, slow to shake cleaning 10min × 3 time；Add secondary antibody in
In reserved 0.5%BSA confining liquid, drop in wet box on PARAFILM film, pvdf membrane being faced down and is laid on after piping and druming mixes
Thereon, it is stored at room temperature 2h；
8) shine: secondary antibody incubation terminates, and film is put into PBST, slow to shake cleaning 10min × 3 time；Equivalent ECL A+B liquid is taken,
Pvdf membrane is faced down on Parafilm film and is laid on 2min thereon, sealed up for safekeeping after draining with preservative film by drop after mixing, with
ChemiDoc Touch chemoluminescence method toning system imagewise development.
In conclusion advantages of the present invention and good effect are as follows: by measuring body itself anti-oxidation stress signal path
The expression of Nrf2/ARE HPAECs in hypoxemia, thus it is speculated that the high expression in pulmonary artery endothelial cell of Nrf2 albumen, and also Radix Astragali is more
Sugar can play anti-oxidative stress by the high expression of enhancing Nrf2 albumen, and function and effect have time and concentration dependant
Property.
COPD disease incidence rises year by year, and patient and social economical burden are increasingly heavy, and astragalus polyose system traditional medicine, valence
Lattice are cheap, and toxic side effect is few, play national medicine strong point, actively for treatment COPD merges pulmonary hypertension, pulmonary heart disease provides newly
Thinking, to play this area Chinese medicine therapeutic features.The present invention is by measuring anti-oxidant product Nrf2 and downstream antioxidase
Expression between different HPAECs groups inquires into Nrf2/AR signal path and the possibility played in oxidative stress occurs in COPD patient
Mechanism；By inquiring into influence of the various concentration astragalus polyose to the Nrf2/ARE signal path of different group HPAECs, inquire into yellow
Whether astragalus polysaccharides antioxidation can effectively delay the oxidation of COPD patient by the expression of enhancing Nrf2/ARE signal path
Stress reaction merges pulmonary hypertension for COPD, pulmonary heart disease seeks effectively preventing approach and establishes experiment basis.
(1) it is logical to specify Nrf2/ARE for the research that first passage expresses Nrf2/ARE signal path in Hypoxic HPAECs
Effect of the road in COPD patient oxidative stress；(2) the spy expression of Nrf2/ARE signal path influenced by different Radix Astragali concentration
Beg for, specify astragalus polyose whether can by enhance Nrf2/ARE signal path expression effectively prevent COPD pulmonary hypertension,
The occurrence and development of pulmonary heart disease merge pulmonary hypertension for COPD, pulmonary heart disease provides new approaches；Astragalus polyose system traditional medicine, valence
Lattice are cheap, and toxic side effect is few, and COPD disease incidence rises year by year, and patient and social economical burden are increasingly heavy, if can be in COPD
It is promoted and applied in patient, to prognosis is improved, mitigates social economical burden, generate great social profit.
COPD hypoxemia is generated reactive oxygen species (ROS) by activation inflammatory cell, is increased patient and is aoxidized burden, and
HPAECs damage is the start-up procedure that Hypoxic Pulmonary Hypertension in Rats occurs, develops.Hypoxemia can lead to barrier function of endothelial cells by
There is dysequilibrium in damage, the vaso-active substance of secretion, and stimulation vascular endothelial cell synthesizes and release ET-l and VEGF increases,
It plays a significant role in Pulmonary vascular remolding and pulmonary hypertension are formed through a variety of ways.Astragalus polyose because have it is anti-oxidant,
It removes the biological activities such as free radical and receives much attention, adjust body response to oxidative stress by many A signal pathways.In showing
Medicine baicalein can increase Nrf2 nuclear translocation and transcriptional activity, enhance body antioxygen directly by the expression of up-regulation Nrf2 albumen
Change ability.So whether Radix Astragali can delay the oxidative stress of COPD anti-by the expression of enhancing Nrf2/ARE signal path
Answer, the present invention inquire into Nrf2/ARE signal path in hypoxemia the expression of HPAECs and with various concentration Effect of APS in Treating act on
The research of correlation, so far both at home and abroad rare report.
The present invention in COPD patient by occurring to the research that HPAECs is expressed in hypoxemia of Nrf2/ARE signal path
Mechanism of action in oxidative stress, to delay, pulmonary hypertension occurs for COPD, pulmonary heart disease provides theoretical foundation；Pass through different Radix Astragalis
Whether the discussion that concentration influences the expression of Nrf2/ARE signal path, have drug concentration dependence with the activation of clear Nrf2/ARE
Property, whether clinical treatment can more effectively delay the occurrence and development of pulmonary hypertension, pulmonary heart disease by adjusting drug dose.
COPD disease incidence rises year by year, and patient and social economical burden are increasingly heavy, and astragalus polyose system traditional medicine, cheap,
Toxic side effect is few, plays national medicine strong point, actively treats COPD merging pulmonary hypertension for this area, pulmonary heart disease provides new think of
Road makes patient be benefited, strives for maximum social benefit to play this area Chinese medicine therapeutic features.
The present invention inquires into body itself anti-oxidation stress signal path Nrf2/ARE in the table of COPD patient HPAECs for the first time
It reaches；Whether astragalus polyose antioxidation can be by the expression of enhancing Nrf2/ARE signal path, to reach effectively anti-for the first time
It controls COPD and merges the occurrence and development of pulmonary hypertension, pulmonary heart disease, and the concentration dependence of curative effect and astragalus polyose is analyzed.
Detailed description of the invention
Fig. 1 be Nrf2/ARE provided in an embodiment of the present invention expressed in hypoxemia and with astragalus polyose protective effect measurement side
Method flow chart.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The present invention is directed to body itself anti-oxidation stress signal path Nrf2/ARE pulmonary artery endothelial cell under hypoxemia
(HPAECs) expression, the mechanism and difference that analysis Nrf2/ARE signal path plays in COPD response to oxidative stress are dense
Astragalus polyose is spent to its regulating and controlling effect；Using adherent method culture HPAECs, by Nrf2/ARE signal path in hypoxemia
HPAECs expression, specifies itself anti-oxidant signal path mechanism of action in COPD patient oxidative stress, to delay COPD
Pulmonary hypertension, pulmonary heart disease provide theoretical foundation；The spy that the expression of Nrf2/ARE signal path is influenced by different Radix Astragali concentration
It begs for, whether there is drug concentration dependence with the activation of clear Nrf2/ARE, to effectively prevent COPD pulmonary hypertension, pulmonary heart disease
Occurrence and development, pulmonary hypertension, pulmonary heart disease may be merged for Effect of APS in Treating COPD and new approaches are provided.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
As shown in Figure 1, Nrf2/ARE provided in an embodiment of the present invention expressed in hypoxemia and with astragalus polyose protective effect
Measuring method the following steps are included:
S101: adherent method culture HPAECs is used, establishes the anoxia model of HPAECs, HPAECs supernatant between measuring different groups
The concentration of Nrf2, SOD, GSH-PX in liquid；
The expression of S102:RT-PCR detection endothelial cell Nrf2mRNA, SODmRNA, GSH-PXmRNA；
S103: western blot determination Nrf2, SOD, GSH-PX protein content.
In a preferred embodiment of the invention, step S101 is specifically included:
3) recovery of HPAECs cell is frozen
1. being immediately placed in 37 DEG C of water-baths, and gently shake cell cryopreservation tube is taken out in liquid nitrogen container, melting it quickly
Change；
2. 1000rpm is centrifuged 5min, is opened after disinfection, suck supernatant with pipette tips；
3. 1640 culture mediums of the cell precipitation with lml containing 10% newborn bovine serum are resuspended, cell suspension moves into 25ml culture
Bottle adds culture solution to total volume to 5ml；
4. be placed in 37 DEG C, 5%CO2, cultivated in saturated humidity cell incubator, for 24 hours after seen under inverted microscope
Examine cell whether adherent growth, replace culture solution；
2) cell passes on
1. 2-3 days replacement culture solutions of the cell of adherent growth are primary, cell, which converges when piece reaches 70-80%, to be passed on；
2. aspirator sucks the liquid in culture dish, 0.25% trypsin solution of 37 DEG C of 1ml preheatings is added,
Microscopically observation is set to after shrinking attached cell, space between cells becomes larger, cell is become round by adherent polygon, removal
Trypsase；
3. being rapidly added 1640/DMEM culture medium, trypsin digestion effect is terminated.Being blown and beaten repeatedly with 3ml liquid-transfering gun makes
Attached cell suspends；
4. cell suspension equivalent is moved into 2-3 25ml culture bottle, it is added into each culture bottle and contains 10% new born bovine
1640 culture medium of serum is placed in 37 DEG C, 5%CO to total amount about 5ml2Saturated humidity cell incubator in continue to cultivate.
The culture solution of HPAECs cell uses 1640 culture mediums, and dual anti-(penicillin concn are as follows: 100U/ml, streptomysin concentration are is added
100μg/ml)；
3) freezing for cell is cultivated:
1. logarithmic growth phase cell, discards culture solution；
2. being added after 0.25% trypsin digestion and containing 10% newborn bovine serum culture medium, blow and beat repeatedly adherent thin
Born of the same parents；
3. cell suspension is moved into the sterile centrifugation tube of 15ml point bottom, 1000rpm is centrifuged 10min, and freezing of preparing is added
Culture solution (contains 10%DMSO), makes the ultimate density 5x10 of cell in frozen stock solution6/ml；
4. being moved into cryopreservation tube with suction pipe, it is put into program cooling freezing storing box, moves into liquid nitrogen container and saves；
4) cobalt chloride HPAECs hypoxia model is established, HPAECs, which is randomly divided into, is divided into five groups: blank control group, hypoxia inducible
Group, hypoxemia+various concentration astragalus polyose (basic, normal, high) intervention group；
1. blank control group: culture plate being placed in room temperature incubator, is added into culture plate and contains 10% blank drug blood
Clear DMEM/F12 culture solution；
2. hypoxia inducible group: culture plate being placed in room temperature incubator, with cobalt chloride effect l2 hours of 0.1%, simultaneously
The DMEM/F12 culture solution for containing 10% blank drug serum is added into culture plate；
3. the low middle and high concentration group of astragalus polyose Contained Serum: small with 0.1% cobalt chloride effect l2 in room temperature incubator
When, the DMEM/F12 containing 1mmol/L, 5mmol/L, 10mmol/L astragalus polyose drug serum is added into culture plate simultaneously respectively
Culture solution；Using adherent method culture HPAECs: HPAECs cell strain being recovered, every other day replacement culture solution is primary, to bottom of bottle
Covering about 80% selects well-grown 3-5 generation cell to be tested with trypsin digestion secondary culture；
5) Nrf2, SOD, GSH-PX in HPAECs supernatant between ELISA measures different groups (according to the explanation of kit)
1. carbonate coating buffer (pH 9.6) of 50mM dissolves cell conditioned medium, make tested factor concentration 10-20
μ g/ml adds 100 holes μ l/ to 96 hole elisa Plates, and 4 DEG C stand overnight.
2. after second day discards coating buffer, being washed 3 times with PBST, it is small that 150 μ l 1%BSA37 DEG C closing 1 is added in every hole
When.
After 3. .PBST is washed 3 times, the supernatant of 100 μ l difference doubling dilution degree is added in every hole, and control sample is added, and 37
DEG C be incubated for 2 hours.
4. after .PBST is washed 5 times, the secondary antibody of the HRP label after 100 μ l dilution is added, 37 DEG C are incubated for 1 hour.
5. after .PBST is washed 5 times, after chromogenic reagent 20min, A405 absorption value is read in microplate reader.
In a preferred embodiment of the invention, step S102 is specifically included:
1) cell total rna extracts
1. sample treatment: after exhausting culture solution, rinsing the cell of adhere-wall culture twice with sterile saline；Cracking: every
Kind sample adds 1ml Trizol reagent to be cracked；
2. layering: 15~30 DEG C are placed 5 minutes, and nucleoprotein complex is promoted to will be completely dissociated, and 0.2ml is added in 1ml Trizol
Chloroform, acutely concussion 15 seconds, 15~30 DEG C of placements, 2~3 minutes (2~8 DEG C), centrifugal rotational speed are no more than 12,000g and are centrifuged 15 points
Supernatant is transferred in new centrifuge tube by clock；
3. RNA precipitate: 0.5ml isopropanol is added by 1ml Trizol reagent in supernatant and mixes, 15~30 DEG C of placements
10 minutes, at 2~8 DEG C, centrifugal rotational speed was no more than 12,000g and is centrifuged 10 minutes abandoning supernatants；
4. RNA is washed: 75% ethanol washing RNA precipitate (75% ethyl alcohol of 1ml is at least added in 1ml Trizol reagent), it can
Be stored in -20 DEG C it is spare；
5. RNA dissolves: oscillation mixes, and centrifugal rotational speed is no more than 7,500g and is centrifuged 5 minutes (2~8 DEG C).Room temperature or vacuum are dry
Dry RNA precipitate is redissolved in DEPC water；
6. RNA concentration and purity testing: detection RNA concentration and purity (AB260B/AB280B ratio)；
2) reverse transcription (RT) step
1. making ice, operates operate on ice if not otherwise indicated below；
2. short term oscillation, centrifugation mix after RNA sample and related reagent thaw；
3. being detected for mRNA level in-site, related reagent is incorporated in thin-walled without in enzyme PCR pipe according to table 1, carries out reverse transcription
Experiment:
1 reverse transcription reaction system of table
4. 37 DEG C of 15min of reverse transcription；85℃ 5sec
3)Real-time PCR
1. GAPDH primer sequence is as follows；
Nrf2:acacggtccacagctcatc (F)；tgtcaatcaaatccatgtcctg(R)；
GSH-PX:accggagaccaggtgatgag(F)；cgatgtcaggctcgatgtca(R)；
SOD:aaagatggtgtggccgatgt(F)；caagccaaacgacttccagc(R)；
GAPDH:ccacccatggcaaattccatggca(F)；tctacacggcaggtcaggtccacc(R)；
2. thin-walled is added without in enzyme PCR pipe in related reagent according to table 2, real-time PCR reaction is carried out
Table 2qPCR reaction system
3. the setting of PCR amplification condition:
Table 3qPCR reaction condition
In a preferred embodiment of the invention, step S103 is specifically included:
Western-blotting detection:
1) cell prepares:
1. endothelial cell is with 5 × 105The density bed board of cells/well after adherent, replaces 1640 culture medium of serum-free；
2. after .24 hours, replacing complete medium, cell is stimulated with various concentration astragalus polyose；
3. after .30 minutes, discarding supernatant liquid, ice PBS is washed three times；
4. extracts total protein with RIPA liquid (containing protease and inhibitors of phosphatases) lytic cell；
5. .BCA method, which surveys 00, determines total protein concentration, sample is added and is mixed with 5 × albumen sample-loading buffer with 4:1 ratio and boils
10 minutes spare；
2) match glue: preparing 10% separation gel；After mixing, separation gel is injected in ready two glass plates crack, and small
The covering of 1ml deionized water is added in the heart in glue surface.After gel natural coagulation (37C about 10~15 minutes), inclination pour out from
Sub- water, and blotted with filter paper.Prepare 5% concentration glue.And it is horizontally inserted into comb in two glass plate cracks, by concentration glue injection point
From glue upper layer, bubble is not stayed.
3) SDS-PAGE electrophoresis: using 70V voltage when bromophenol blue moves to separation gel after change 120V voltage to bromophenol blue move
It when to from bottom, cuts off the power, stops electrophoresis.
4) transferring film: electrophoresis terminates, and glue is peeled, and cuts spacer gel, carries out front and back sides in the upper right corner and marks, buffers in transferring film
15min is impregnated in liquid；It measures and cuts pvdf membrane one of the same size and open, also in prime marks and Whatman 3MM filter paper
6；Successively put sponge, 3 Whatman filter paper, pvdf membrane, SDS-PAGE glue, 3 Whatman filter paper well from anode to cathode
And sponge, it is inserted into transferring film slot, 310mA constant current, 100min；Transferring film finishes, and removes pvdf membrane, the dyeing of 1 × Ponceaux dyeing liquor
Transferring film effect is observed, PBST washes away dye liquor；
5) it closes: the pvdf membrane shifted is placed in 0.5%BSA confining liquid, room temperature is slow to shake 2h；
6) primary antibody is incubated for: after closing, film is put into PBST, slow to shake cleaning 10min × 3 time；Add primary antibody in reserving it
In 0.5%BSA confining liquid, after piping and druming mixes, drop in wet box on PARAFILM film, pvdf membrane being faced down and is laid on thereon, and 4
DEG C overnight；
7) secondary antibody is incubated for: after primary antibody is incubated for, film is put into PBST, slow to shake cleaning 10min × 3 time；Add secondary antibody in
In reserved 0.5%BSA confining liquid, drop in wet box on Parafilm film, pvdf membrane being faced down and is laid on after piping and druming mixes
Thereon, it is stored at room temperature 2h；
8) shine: secondary antibody incubation terminates, and film is put into PBST, slow to shake cleaning 10min × 3 time；Equivalent ECL A+B liquid is taken,
Pvdf membrane is faced down on Parafilm film and is laid on 2min thereon, sealed up for safekeeping after draining with preservative film by drop after mixing, with
ChemiDoc Touch chemoluminescence method toning system imagewise development.
Both at home and abroad multiple studies have shown that Keapl/Nrf2-ARE signal transduction pathway takes part in nervous system, endocrine system
System, the reaction of itself anti-oxidation stress of tumour system generate II phase detoxication enzyme by induction body and the high of anti-oxidant albumen are expressed,
The oxidative damage of antagonism tissue.Sulforaphane can activate Nrf2 signal path, up-regulation antioxidant enzyme and quinone oxidation downstream
The expression of the enzymes such as reductase 1 (NQO-1), it is now recognized that Nrf2 activation is the committed step that cell resists oxidative stress, but still
Without research shows that participate in COPD response to oxidative stress, COPD merge pulmonary hypertension, pulmonary heart disease pathophysiological change in,
The conditions associated with hypoxia damage of HPAECs plays a significant role, and by the regulation to Keap1/Nrf2/ARE access, it is likely to become prevention
With the important method for the treatment of COPD complication.(1): inquiring into body itself anti-oxidation stress signal path Nrf2/ARE for the first time low
The expression of people HPAECs when oxygen；(2) whether: studying astragalus polyose antioxidation for the first time can be by enhancing Nrf2/ARE signal
The expression of access, to effectively prevent the occurrence and development that COPD merges pulmonary hypertension, pulmonary heart disease, and to curative effect and astragalus polyose
Concentration dependence is analyzed.The present invention is by measurement body itself anti-oxidation stress signal path Nrf2/ARE in hypoxemia
The expression of HPAECs, thus it is speculated that the high expression in pulmonary artery endothelial cell of Nrf2 albumen, and also astragalus polyose can pass through enhancing
The high expression of Nrf2 albumen plays anti-oxidative stress, and function and effect have time-and concentration-dependent, effectively prolong COPD
Merge the course of disease of pulmonary hypertension, pulmonary heart disease.
COPD disease incidence rises year by year, and patient and social economical burden are increasingly heavy, and astragalus polyose system traditional medicine, valence
Lattice are cheap, and toxic side effect is few, play national medicine strong point, actively treat COPD for this area and merge pulmonary hypertension, pulmonary heart disease
New approaches are provided, to play this area Chinese medicine therapeutic features.(1) by measuring anti-oxidant product Nrf2 and anti-oxidant downstream
The possibility machine played in oxidative stress occurs in COPD patient for expression of the enzyme between different HPAECs groups, Nrf2/ARE signal path
System；(2) it by inquiring into influence of the various concentration astragalus polyose to the Nrf2/ARE signal path of different group HPAECs, inquires into yellow
Whether astragalus polysaccharides antioxidation can effectively delay the oxidation of COPD patient by the expression of enhancing Nrf2/ARE signal path
Stress reaction merges pulmonary hypertension for COPD, pulmonary heart disease seeks effectively preventing approach and establishes experiment basis.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (4)
1. a kind of Nrf2/ARE expressed in hypoxemia and with astragalus polyose protective effect measuring method, which is characterized in that it is described
Nrf2/ARE is expressed in hypoxemia and is included: with astragalus polyose protective effect measuring method
The first step establishes the anoxia model of HPAECs using adherent method culture HPAECs, HPAECs supernatant between measuring different groups
The concentration of middle Nrf2, SOD, GSH-PX；
Second step, RT-PCR detect endothelial cell Nrf2mRNA, SOD mRNA, GSH-PXmRNA, expression；
Third step, western blot determination Nrf2, SOD, GSH-PX protein content.
2. Nrf2/ARE as described in claim 1 expressed in hypoxemia and with astragalus polyose protective effect measuring method, it is special
Sign is that the first step specifically includes:
1) recovery of HPAECs cell is frozen
1. being immediately placed in 37 DEG C of water-baths, and gently shake cell cryopreservation tube is taken out in liquid nitrogen container, making its fast melt；
2. 1000rpm is centrifuged 5min, is opened after disinfection, suck supernatant with pipette tips；
3. 1640 culture mediums of the cell precipitation with lml containing 10% newborn bovine serum are resuspended, cell suspension moves into 25ml culture bottle, adds
Culture solution is to total volume to 5ml；
4. being placed in 37 DEG C, 5%CO2, cultivated in saturated humidity cell incubator, for 24 hours after observe cell under inverted microscope
Whether adherent growth, replace culture solution；
2) cell passes on
1. 2-3 days replacement culture solutions of the cell of adherent growth are primary, cell, which converges when piece reaches 70-80%, to be passed on；
2. aspirator sucks the liquid in culture dish, 0.25% trypsin solution of 37 DEG C of 1ml preheatings is added, is inverted aobvious
After observing that attached cell shrinks, space between cells becomes larger, cell is become round by adherent polygon under micro mirror, pancreas egg is removed
White enzyme；
3. being rapidly added 1640/DMEM culture medium, trypsin digestion effect is terminated.Blown and beaten repeatedly with 3ml liquid-transfering gun make it is adherent
Cell suspends；
4. cell suspension equivalent is moved into 2-3 25ml culture bottle, it is added into each culture bottle and contains 10% newborn bovine serum
1640 culture mediums are placed in 37 DEG C, 5%CO to total amount about 5ml2Saturated humidity cell incubator in continue to cultivate.HPAECs is thin
The culture solution of born of the same parents uses 1640 culture mediums, and dual anti-, penicillin concn is added are as follows: 100U/ml, streptomysin concentration are 100 μ g/ml；
3) freezing for cell is cultivated:
1. logarithmic growth phase cell, discards culture solution；
2. being added after 0.25% trypsin digestion and containing 10% newborn bovine serum culture medium, blow and beat attached cell repeatedly；
3. by cell suspension move into 15ml point bottom sterile centrifugation tube in, 1000rpm be centrifuged 10min, be added prepare freeze culture
Liquid contains 10%DMSO, makes the ultimate density 5x10 of cell in frozen stock solution6/ml；
4. being moved into cryopreservation tube with suction pipe, it is put into program cooling freezing storing box, moves into liquid nitrogen container and saves；
4) cobalt chloride HPAECs hypoxia model is established, HPAECs is randomly divided into five groups: blank control group, hypoxia inducible group, hypoxemia+
The basic, normal, high intervention group of various concentration astragalus polyose；
1. blank control group: culture plate being placed in room temperature incubator, is added into culture plate containing 10% blank drug serum
DMEM/F12 culture solution；
2. hypoxia inducible group: culture plate being placed in room temperature incubator, is acted on l2 hours with 0.1% cobalt chloride, while to training
Support the DMEM/F12 culture solution for being added in plate and containing 10% blank drug serum；
3. the low middle and high concentration group of astragalus polyose Contained Serum: in room temperature incubator, acted on l2 hours with 0.1% cobalt chloride, point
The DMEM/F12 culture containing 1mmol/L, 5mmol/L, 10mmol/L astragalus polyose drug serum is not added into culture plate not simultaneously
Liquid；Using adherent method culture HPAECs: HPAECs cell strain being recovered, every other day replacement culture solution is primary, covers to bottom of bottle
About 80%, with trypsin digestion secondary culture, well-grown 3-5 generation cell is selected to be tested；
5) Nrf2, SOD, GSH-PX in HPAECs supernatant between ELISA measures different groups；
1. is coated with buffer solution cell conditioned medium with the carbonate of 50mM, makes tested factor concentration 10-20 μ g/ml, add 100 μ
To 96 hole elisa Plates, 4 DEG C are stood overnight in the hole l/；
2. after second day discards coating buffer, being washed 3 times with PBST, every hole is added 37 DEG C of 150 μ l 1%BSA and closes 1 hour；
3. after .PBST is washed 3 times, the supernatant of 100 μ l difference doubling dilution degree is added in every hole, and control sample is added, and 37 DEG C incubate
It educates 2 hours；
4. after .PBST is washed 5 times, the secondary antibody of the HRP label after 100 μ l dilution is added, 37 DEG C are incubated for 1 hour；
5. after .PBST is washed 5 times, after chromogenic reagent 20min, A405 absorption value is read in microplate reader.
3. Nrf2/ARE as described in claim 1 expressed in hypoxemia and with astragalus polyose protective effect measuring method, it is special
Sign is that the second step specifically includes:
1) cell total rna extracts
1. sample treatment: after exhausting culture solution, rinsing the cell of adhere-wall culture twice with sterile saline；Cracking: every kind of sample
Product add 1ml Trizol reagent to be cracked；
2. layering: 15~30 DEG C are placed 5 minutes, and nucleoprotein complex is promoted to will be completely dissociated, and 0.2ml chlorine is added in 1ml Trizol
It is imitative, it acutely shakes 15 seconds, 15~30 DEG C are placed 2~3 minutes, and centrifugal rotational speed is no more than 12,000g and is centrifuged 15 minutes, by supernatant
It is transferred in new centrifuge tube；
3. RNA precipitate: 0.5ml isopropanol is added by 1ml Trizol reagent in supernatant and mixes, 15~30 DEG C are placed 10 points
Clock, at 2~8 DEG C, centrifugal rotational speed is no more than 12,000g and is centrifuged 10 minutes abandoning supernatants；
4. RNA is washed: 75% ethyl alcohol of 1ml is at least added in 75% ethanol washing RNA precipitate, 1ml Trizol reagent, be stored in-
20 DEG C spare；
5. RNA dissolve: oscillation mix, centrifugal rotational speed be no more than 7,500g be centrifuged 5 minutes 2~8 DEG C；Room temperature or vacuum drying RNA
Precipitating, is redissolved in DEPC water；
6. RNA concentration and purity testing: detection RNA concentration and purity；
2) reverse transcription step
1. making ice, operates operate on ice if not otherwise indicated below；
2. short term oscillation, centrifugation mix after RNA sample and related reagent thaw；
3. being detected for mRNA level in-site, related reagent is incorporated in thin-walled without in enzyme PCR pipe according to table 1, carries out reverse transcription experiment:
4. 37 DEG C of 15min of reverse transcription；85℃5sec；
3)Real-time PCR
1. GAPDH primer sequence is as follows；
Nrf2:acacggtccacagctcatc (F)；tgtcaatcaaatccatgtcctg(R)；
GSH-PX:accggagaccaggtgatgag(F)；cgatgtcaggctcgatgtca(R)；
SOD:aaagatggtgtggccgatgt(F)；caagccaaacgacttccagc(R)；
GAPDH:ccacccatggcaaattccatggca(F)；tctacacggcaggtcaggtccacc(R)；
2. thin-walled is added without in enzyme PCR pipe in related reagent, real-time PCR reaction is carried out；
3. the setting of PCR amplification condition.
4. Nrf2/ARE as described in claim 1 expressed in hypoxemia and with astragalus polyose protective effect measuring method, it is special
Sign is that the third step specifically includes:
Western-blotting detection:
1) cell prepares:
1. endothelial cell is with 5 × 105The density bed board of cells/well after adherent, replaces 1640 culture medium of serum-free；
2. after .24 hours, replacing complete medium, cell is stimulated with various concentration astragalus polyose；
3. after .30 minutes, discarding supernatant liquid, ice PBS is washed three times；
4. extracts total protein with RIPA liquid lytic cell；
5. .BCA method, which surveys 00, determines total protein concentration, sample is added and is mixed with 5 × albumen sample-loading buffer with 4:1 ratio and boils 10 points
Clock is spare；
2) match glue: preparing 10% separation gel；After mixing, separation gel is injected in ready two glass plates crack, and carefully exists
The covering of 1ml deionized water is added in glue surface；After gel natural coagulation, deionized water is poured out in inclination, and is blotted with filter paper；It prepares
5% concentration glue；And it is horizontally inserted into comb in two glass plate cracks, concentration glue is injected into separation gel upper layer, not stay bubble；
3) SDS-PAGE electrophoresis: using 70V voltage when bromophenol blue moves to separation gel after change 120V voltage to bromophenol blue move on to from
It when bottom, cuts off the power, stops electrophoresis；
4) transferring film: electrophoresis terminates, and glue is peeled, and cuts spacer gel, carries out front and back sides in the upper right corner and marks, in transferring film buffer
Impregnate 15min；It measures and cuts pvdf membrane one of the same size and open, also opened in prime marks and Whatman 3MM filter paper 6；
Successively put sponge, 3 Whatman filter paper, pvdf membrane, SDS-PAGE glue, 3 Whatman filter paper and sea well from anode to cathode
Silk floss is inserted into transferring film slot, 310mA constant current, 100min；Transferring film finishes, and removes pvdf membrane, the dyeing observation of 1 × Ponceaux dyeing liquor
Transferring film effect, PBST wash away dye liquor；
5) it closes: the pvdf membrane shifted is placed in 0.5%BSA confining liquid, room temperature is slow to shake 2h；
6) primary antibody is incubated for: after closing, film is put into PBST, slow to shake cleaning 10min × 3 time；Add primary antibody in reserved 0.5%
In BSA confining liquid, after piping and druming mixes, drop in wet box on PARAFILM film, pvdf membrane being faced down and is laid on thereon, 4 DEG C of mistakes
Night；
7) secondary antibody is incubated for: after primary antibody is incubated for, film is put into PBST, slow to shake cleaning 10min × 3 time；Secondary antibody is added in reserved
0.5%BSA confining liquid in, after piping and druming mixes drop in wet box on Parafilm film, pvdf membrane being faced down and is laid on thereon,
It is stored at room temperature 2h；
8) shine: secondary antibody incubation terminates, and film is put into PBST, slow to shake cleaning 10min × 3 time；Equivalent ECL A+B liquid is taken, is mixed
After drip on Parafilm film, pvdf membrane is faced down and is laid on 2min thereon, is sealed up for safekeeping after draining with preservative film, with ChemiDoc
Touch chemoluminescence method toning system imagewise development.
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Citations (2)
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| US20110250300A1 (en) * | 2005-07-01 | 2011-10-13 | The Johns Hopkins University | Compositions and methods for the treatment or prevention of disorders relating to oxidative stress |
| CN108159223A (en) * | 2017-12-13 | 2018-06-15 | 新疆大学 | Chufa extractive of general flavone, activated monomer orientoside and its extracting method and application |
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2019
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110250300A1 (en) * | 2005-07-01 | 2011-10-13 | The Johns Hopkins University | Compositions and methods for the treatment or prevention of disorders relating to oxidative stress |
| CN108159223A (en) * | 2017-12-13 | 2018-06-15 | 新疆大学 | Chufa extractive of general flavone, activated monomer orientoside and its extracting method and application |
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| JIACUI ZHANG等: "Metformin reverses the resistance mechanism of lung adenocarcinoma cells that knocks down the Nrf2 gene", 《ONCOLOGY LETTERS》 * |
| SIMONA ADESSO等: "Astragalus membranaceus Extract Attenuates Inflammation and Oxidative Stress in Intestinal Epithelial Cells via NF-κB Activation and Nrf2 Response", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》 * |
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| 王芳等: "基于Keap1-Nrf2/ARE信号传导通路探讨黄芪多糖改善干燥综合征模型大鼠心功能的机制", 《中国中西医结合杂志》 * |
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