CN102608089B - Detection method for isolated perfusion heart active oxygen and mitochondrial membrane potential - Google Patents
Detection method for isolated perfusion heart active oxygen and mitochondrial membrane potential Download PDFInfo
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Abstract
The invention relates to a detection method for isolated perfusion heart active oxygen and mitochondrial membrane potential, which comprises the following steps of: establishing an isolated perfusion heart ischemia reperfusion model; realizing correlation of an isolated heart perfusion system, a probe loading device and a laser scanning confocal microscope; further carrying out real-time loading and microscope imaging on a fluorescence probe of the active oxygen and mitochondrial membrane potential; and finally, through image processing and data analysis, the method can be used for real-time and positioning measurement of heart ROS (reactive oxygen species) and mitochondrial membrane potential in various isolated heart models taking acute overload, ischemia reperfusion and the like as representatives under a stable perfusion state. The detection method has the characteristics of directness, accuracy, visibility, time and space positioning, and synchronized measurement of beating cardiac metabolism and function, and has the advantages of low cost, high efficiency, and good reliability and repeatability.
Description
Technical field
The present invention relates to the detection technique field of intracellular reactive chalcogen and mitochondrial membrane potential is and in particular to one kind is in vitro
Heart perfusion activity chalcogen and the detection method of mitochondrial membrane potential.
Technical background
Active chalcogen (Reactive Oxygen Species, ROS) is the oxygen-derived free radicals being formed in organism oxidation reaction
With the derivant of non-free radical oxygen, wherein with superoxide anion, hydrogen peroxide be modal, active the strongest.The oxygen that ROS causes
Change stress be a series of important incitant of cardiovascular disease, therefore accurate detection cell ROS content and closely related therewith
Mitochondrial membrane potential, for major cardiovascular disease research with prevent and treat significant.
Because the life-span of biological internal active oxygen is extremely short, Css is extremely low, extremely difficult to its Accurate Determining.The heart at present
The detection method of flesh ROS is broadly divided into direct detection and indirect detection two big class.Direct detecting method includes electron spin resonance
Method, high performance liquid chromatography, chemoluminescence method and fluorescence probe method etc..Wherein first two has high demands to instrument and equipment, and cost
Height, efficiency is low, and flow process is complicated；Chemoluminescence method relative ease, but credible result degree is relatively low, and cannot determine with space real-time tracking
Position.Using fluorescent probe labelling, can be detected in conjunction with flow cytometer and fluorescence microscopy, wherein fluorescent probe combines
Confocal microscopic image technology is the currently the only detection side enabling living cells and in-house ROS " real-time, visible, quantitative "
Method.However, being only limitted to cell, ice on drawing materials so far Bao Kuo the above-mentioned ROS direct detecting method including fluorescence probe method
Freeze tissue or tissue homogenate.Wherein, the detection of cell of drawing materials often cannot accurately reflect internal homergy and functional statuses
Under ROS level.For example：The myocardial cell system of culture or the primary cardiomyocytes of separation and Culture, its cell phenotype, character and work(
Feature all can there occurs very big change compared with body state；Even the myocardial cell of acute isolation, also unavoidably because of acute point
From during adjoint cell injury and interference is produced to the measurement of ROS.And for frozen tissue, short due to the ROS half-life
Temporarily, the susceptiveness making ROS detect and specificity are had a greatly reduced quality by frozen-thaw process.The such as cell or tissue homogenate of other method of drawing material
Liquid, homogenization process will bring cell injury and/or ROS to be quenched, and substantially reduce accuracy and the susceptiveness of ROS detection.
The Indirect Detecting Method often observation based on the terminal effect to ROS mediated cell peroxide injury, such as utilizes 8-
The method detection DNA oxidative damage of OHdG antibody mediated immunity histochemical staining, by the evaluation of measuring lipid mistake of malonaldehyde (MDA) content
The methods such as oxidative damage degree.The simply accumulative effect observed due to Indirect Detecting Method, lacks specificity it is impossible to ROS's
Generation carries out time and space orientation, and cannot be distinguished by specific ROS species.
With respect to the detection method from cell and frozen tissue sampling, isolated heart perfusion technology had both remained organ dysfunction
With the globality of metabolism, eliminate nerve, the interference of humoral factor under concrete conditions in the establishment of a specific crime again, can be used for studying hypoxic-ischemic, heart
The impact to heart for the multiple intervention condition such as acute overload, medicine, toxin.Perfused isolated heart experimental system is that one kind is used for
, under manual control condition, various factors (as nutrient substance, medicine etc.) is to cardiomotility for research mammal isolated heart
Impact.The signals such as the ventricular pressure of isolated heart activity, electrocardio with the collection of multiple tracks bio signal analysis system, process and can be analyzed.
Therefore detect ROS level in isolated perfused heart, not only can realize that ROS is " real-time, visible, quantitative " to be detected moreover it is possible to and heart
Contractile function, metabolism state synchronous recording, and then carry out ROS and heart work(in multiple physiology, pathological process and intervening measure
Energy, the Real-Time Evaluation of metabolism.
Thing followed problem is how ROS fluorescent probe technique to be applied to Perfused isolated heart system.Existing at present
Multiple commercializations and widely used ROS probe, can be used for dissimilar ROS and the ROS labelling of different ultrastructure distribution.
However, and not all ROS probe can obtain good labelling effect can be in laser copolymerization in isolated perfused heart tissue
Real-time, positioning requirement is reached in burnt imaging process.We are to intracellular O2 -Probe dihydro second pyridine
In (Dihydroethidium, DHE) and its application of derivant find, DHE in experimentation except with O2 -Reaction generates oxygen
Change outside product oxidation second pyridine, also can produce a kind of non-specific oxidation product ethidium, and both absorbing wavelength are very approximate.
Therefore under traditional 514nm excitation wavelength, the specificity of DHE and non-specific oxidation product cannot be distinguished by.It is therefore to reject bromine
Second this nonspecific products of non-pyridine, traditional method need to carry out accurate quantification with high performance liquid chromatography.Equally, Mitochondrially targeted O2 -
Probe Mitosox red, as the derivant of DHE mitochondria positioning, there is also O2 -Specific problem.It was found that it is logical
Cross and compare two kinds of different excitation wavelengths (i.e. 405nm and 514nm excitation wavelength) correspondence 580nm absorbing wavelength, can specificity inspection
Survey DHE and Mitosox red and O2 -Effect product, i.e. O2 -Specific marker.Additionally, the art of this patent is by visiting to different
The analysis of pin optical characteristics, adopts three carrying methods first, not only can greatly reduce experiment sample amount, also can be in same sample
Relation between middle relative analyses different measuring target.
Another needs the problem solving to be, heart continuous pulsation and will simultaneously maintain normal filling during imaging acquisition
Stream, experimental apparatus and perfusion system also need to improve accordingly to ensure effective collection of image.Because of the heart in image acquisition process
Dirty contraction causes artifact, adopt in the art of this patent in perfusate plus with muscle fiber inhibitor Blebbistatin (10mM) with
Reduce heart rate, image quality can be effectively improved.
Content of the invention
In order to overcome the shortcoming of above-mentioned prior art, it is an object of the invention to provide a kind of isolated perfused heart active oxygen
Race and the detection method of mitochondrial membrane potential, are related to probe selection, labeling method, the setting of image-forming condition, image acquisition parameter
The key link such as selection, have the characteristics that in real time, in situ, can position, quantitative, and low cost, efficiency high, credibility and repetition
Property is good.
For reaching above-mentioned purpose, the technical solution used in the present invention is：
A kind of isolated perfused heart activity chalcogen and the detection method of mitochondrial membrane potential, comprise the following steps：
The first step, the foundation of Isolated Heart perfusion Model, take small-sized mammalian to open rapidly breast after heparinization, anesthesia and take
Go out heart and be fixed on Langendorff perfusion system, through aorta perfusion retrogradely with 95%O2+ 5%CO2Mixed gas saturation
K-H liquid (37 DEG C, pH 7.4, mmol/L)：NaCl 115, NaHCO325, glucose 11, KCl 4.0, CaCl21.5,
MgSO40.9, KH2PO40.9, for real-time measurement cardiac systolic function, cut an opening in left atrium pulmonary vein porch, by one even
The sacculus being connected to pressure transducer inserts left ventricle through left atrium, is filled with deionized water until reaching after being completely exhausted out air in sacculus
To the BDP of 5-10mmHg, by multi-path physiology record system continuous Real-time Collection left ventricular pressure index, heart rate HR, left room
Systolic pressure LVSP, left ventricular end diastolic presssure LVEDP, left chamber pressure change maximum rate ± LV dP/dtmaxData, by computer from
Move to record and calculate process and carry out Evaluation on Cardiac Function,
Second step, isolated perfusion system is associated with probe load device and microscopical instrument, using Zeiss LSM 510
Laser Scanning Confocal Microscope, the high optical glass ware of object stage center placement 3-5cm diameter, 1-1.5cm, by heart perfusion left ventricular apex
Portion is affixed on glass dish, perfusion pipe end clamp is fixed it is ensured that heart stabilizer is affixed on glass dish avoids displacement, glass dish
A drainage tube placed by side wall, through peristaltic pump drive can automatic jet heart perfusion effluent, the load of fluorescent probe utilizes micro-
Amount pump installation, the probe concentrated solution being placed in syringe at the uniform velocity pumps into through micro pump, and syringe connects threeway and the heart by flexible pipe
The perfusion terminal of dirty perfusion device is connected, and is adjusted and is controlled fluorescent probe concentrated solution by the opening and closing and micro pump pump speed of threeway
Input opportunity and input speed,
3rd step, fluorescent probe loads, and after isolated heart stablizes 15-20 minute under the basic perfusion state, gives to be directed to
O2 -、H2O2, mitochondrion specificity O2 -, and grain wire body transmembrane potential fluorescent probe：DHE(10-5Mol/L), DCFDA (5X10- 5Mol/L), MitoSOX Red (2.5X10-6Mol/L), TMRE (5X10-6Mol/L), give simultaneously DAPI (1 μ g/ml) and/or
MitoGreen(5X10-7Mol/L) lining dye nucleus and mitochondrion, fluorescent probe loading concentrations and speed are passed through to arrange micro pump
Pump into speed controlling, micro pump pumps into speed=probe face liquid concentration/probe concentrated solution concentration * 100%, using bigeminy or three
Connection carrying method, used when fluorescent probe specificity used, working concentration and fluorescence microscope imaging excite/absorbing wavelength sees below
Table,
Note：In table, fluorescent probe used is all from Molecular Probes,TMInvitrogen
4th step, implements Experiment intervention means, including setting up isolated heart ischemia-reperfusion, the acute overload of heart, the heart
Dirty frequency modulation and administration model,
5th step, laser confocal microscope is imaged, at 30 μm of centroid adventitia myocardium group of fluorescent collecting flat focus
In knitting, as caused artifact because of heartbeat excessive velocities in image acquisition process, then add in perfusate and use muscle fiber inhibitor
Blebbistatin (5-10mM), to reduce heart rate, loads after 10-15 minute after fluorescent probe, to fluorescence signal in heart perfusion
Continuously scanned, setting sweep interval is 5 minutes, respectively to excite/absorbing wavelength corresponding to fluorescent probe used
It is imaged under 63 times of oil mirrors, the every frame of scanning speed 0.7 second,
6th step, image procossing and analysis, ImageJ computed in software fluorescence signal intensity, wherein endochylema O2 -Level DHE
Fluorescence intensity ratio under the different absorbing wavelength of 405nm with 514nm represents, mitochondrion O2 -Level MitoSox in 405nm and
Fluorescence intensity ratio under 514nm difference absorbing wavelength represents.Endochylema O2 -、H2O2, mitochondrion specificity O2 -With mitochondrial membrane electricity
Position fluorescence signal does weight with the nucleus representated by DAPI/Vybrant Ruby and MitoGreen and mitochondrial signal respectively.
The foundation by Isolated Heart perfusion Model for the present invention, realizes probe load technology and laser confocal microscope
Device is associated with Perfused isolated heart system, carry out active chalcogen and mitochondrial membrane potential the real time load of fluorescent probe and
Laser confocal microscope is imaged, and finally by the analysis of image procossing data, surpasses to stablizing under perfusion state and with acute
Load, ischemia-reperfusion etc. be the dirty ROS of multiple isolated heart model center of representative and mitochondrial membrane potential carry out in real time, position
Measurement.
Brief description
Fig. 1 is O in myocardial cell during the normal perfusion of rabbit isolated heart of the embodiment of the present invention 12 -Laser co-focusing imaging
Picture.
Fig. 2 is myocardial cell mitochondrion during the Isolated Perfused Rat heart calcium acute over loading of load of the embodiment of the present invention 2
O2 -Laser co-focusing imaging picture.
Fig. 3 is H in myocardial cell in the mice isolated perfused heart Ischemia-Reperfusion Injury of the embodiment of the present invention 32O2With
The laser co-focusing imaging picture of mitochondrial membrane potential.
Specific embodiment
With reference to embodiment, the present invention is described in detail.
Embodiment one
A kind of isolated perfused heart activity chalcogen and the detection method of mitochondrial membrane potential, specially a kind of rabbit isolated heart
O in myocardial cell during normal perfusion2 -Real-time in-situ detection method, comprise the following steps：
The first step, the foundation of Isolated Heart perfusion Model, pentobarbital sodium (50mg/kg, i.p.) anesthesia heparinization
(500IU/kg, i.v.) rabbit, opens rapidly breast and takes out heart and be fixed on Langendorff perfusion system, drive in the wrong direction through aorta and fill
Stream is with 95%O2+ 5%CO2The K-H liquid (37 DEG C, pH 7.4, mmol/L) of mixed gas saturation：NaCl 115, NaHCO325,
Glucose11, KCl 4.0, CaCl21.5, MgSO40.9, KH2PO40.9, measure cardiac systolic function for real-time, in left atrium
An opening is cut in pulmonary vein porch, a sacculus being connected to pressure transducer is inserted left ventricle through left atrium, is completely exhausted out ball
It is filled with deionized water until reaching the BDP of 5-10mmHg after intracapsular air, continuously real-time by multi-path physiology record system
Collection left ventricular pressure index, heart rate HR, left ventricular systolic pressure LVSP, left ventricular end diastolic presssure LVEDP, left chamber pressure change maximum rate ±
LV dP/dtmaxData, automatically recorded and calculated by computer process and carry out Evaluation on Cardiac Function, treat that heart rate and contractile function are steady
The perfusate perfused hearts being added with muscle fiber inhibitor Blebbistatin (5mM) are used instead to reduce heart rate after fixed,
Second step, isolated perfusion system is associated with probe load device and microscopical instrument, using Zeiss LSM 510
Laser Scanning Confocal Microscope, the high optical glass ware of object stage center placement 5cm diameter, 1.5cm, heart perfusion left ventricular apex portion is pasted
On glass dish, perfusion pipe end clamp is fixed it is ensured that heart stabilizer is affixed on glass dish avoids displacement, glass dish side wall
Place a drainage tube, through peristaltic pump drive can automatic jet heart perfusion effluent.The load of fluorescent probe mainly utilizes micro-
Amount pump installation, the 50X probe concentrated solution being placed in syringe at the uniform velocity pumps into through micro pump, syringe pass through flexible pipe connect threeway and
The perfusion terminal of cardiac perfusion device is connected, and is adjusted and is controlled fluorescent probe concentrated solution by the opening and closing and micro pump pump speed of threeway
Input opportunity and input speed,
3rd step, fluorescent probe loads, and after isolated heart stablizes 15 minutes under the basic perfusion state, gives O2 -Fluorescence is visited
Pin DHE (5X10-6Mol/L), use Vybrant Ruby (2.5X10 simultaneously-6Mol/L) lining dye nucleus, fluorescent probe load is dense
Degree and speed pump into speed controlling by arranging micro pump, and micro pump pumps into speed and is set to heart coronary artery outflow flow velocity
2%, that is,：If rabbit isolated heart coronary outflow flow velocity be 20ml/min, micro pump pump into speed be set to (20ml/min ×
2%=0.4ml/min, i.e. 24ml/h), fluorescent probe concentration used, compound method and fluorescence microscope imaging when used excite/
Absorbing wavelength is as follows,
Note：In table, fluorescent probe used is all from Molecular Probes,TMInvitrogen
4th step, implements Experiment intervention means, including setting up isolated heart ischemia-reperfusion, the acute overload of heart, the heart
Dirty frequency modulation and administration model,
5th step, laser confocal microscope is imaged, at 30 μm of centroid adventitia myocardium group of fluorescent collecting flat focus
In knitting, as caused artifact because of heartbeat excessive velocities in image acquisition process, then add in perfusate and use muscle fiber inhibitor
Blebbistatin (5-10mM), to reduce heart rate, after fluorescent probe loads 10 minutes, enters to fluorescence signal in heart perfusion
Row is continuous to be scanned, fluorescent collecting flat focus in the cardiac muscular tissue at about 30 μm of centroid adventitia, respectively with 514/580,405/
580th, 633/670 excite/absorbing wavelength is imaged under 63 times of oil mirrors, the every frame of scanning speed 0.7 second,
6th step, image procossing and analysis, ImageJ computed in software fluorescence signal intensity, wherein intracellular O2 -Level is used
Fluorescence intensity ratio under the different absorbing wavelength of 405nm with 514nm for the DHE represents, and with thin representated by Vybrant Ruby
Karyon signal does weight.
Embodiment two
A kind of isolated perfused heart activity chalcogen and the detection method of mitochondrial membrane potential, specially a kind of isolated rat fills
Myocardial cell mitochondrion O when stream heart calcium loads acute over loading2 -Real-time in-situ detection method, comprise the following steps：
The first step, the foundation of isolated rat heart perfusion model, pentobarbital sodium (50mg/kg, i.p.) anesthesia heparin
Change (500IU/kg, i.v.) rat, open rapidly breast and take out heart and be fixed on Langendorff perfusion system, inverse through aorta
Row perfusion is with 95%O2+ 5%CO2The K-H liquid (37 DEG C, pH 7.4, mmol/L) of mixed gas saturation：NaCl 115,
NaHCO325, glucose 11, KCl 4.0, CaCl21.5, MgSO40.9, KH2PO40.9, measure cardiac systolic function for real-time,
Cut an opening in left atrium pulmonary vein porch, a sacculus being connected to pressure transducer is inserted left ventricle through left atrium, complete
It is filled with deionized water until reaching the BDP of 5-10mmHg, by multi-path physiology record system after air in full discharge sacculus
Continuous Real-time Collection left ventricular pressure index, heart rate HR, left ventricular systolic pressure LVSP, left ventricular end diastolic presssure LVEDP, left chamber pressure change
Big speed ± LV dP/dtmaxData, automatically recorded and calculated by computer process and carry out Evaluation on Cardiac Function, treat heart rate and receipts
The perfusate perfused hearts being added with muscle fiber inhibitor Blebbistatin (10mM) are used instead to reduce the heart after contracting function-stable
Rate,
Second step, isolated perfusion system is associated with probe load device and microscopical instrument, using Zeiss LSM 510
Laser Scanning Confocal Microscope, the high optical glass ware of object stage center placement 3cm diameter, 1cm, heart perfusion left ventricular apex portion is affixed on
On glass dish, perfusion pipe end clamp is fixed it is ensured that heart stabilizer is affixed on glass dish avoids displacement, glass dish side wall is put
Put a drainage tube, through peristaltic pump drive can automatic jet heart perfusion effluent, the load of fluorescent probe utilizes micro pump to fill
Put, the 20X probe concentrated solution being placed in syringe at the uniform velocity pumps into through micro pump, syringe is connect threeway by flexible pipe and filled with heart
The perfusion terminal of stream device is connected, and adjusts, by the opening and closing and micro pump pump speed of threeway, the input controlling fluorescent probe concentrated solution
Opportunity and input speed,
3rd step, fluorescent probe loads, and after isolated heart stablizes 15 minutes under the basic perfusion state, gives O2 -Fluorescence is visited
Pin DHE (5X10-6Mol/L), use Vybrant Ruby (2.5X10 simultaneously-6Mol/L) lining dye nucleus, fluorescent probe load is dense
Degree and speed pump into speed controlling by arranging micro pump, and micro pump pumps into speed and is set to heart coronary artery outflow flow velocity
5%, that is,：If isolated rat heart coronary outflow flow velocity be 8ml/min, micro pump pump into speed be set to (8ml/min ×
5%=0.4ml/min, i.e. 24ml/h), fluorescent probe concentration used, compound method and fluorescence microscope imaging when used excite/
Absorbing wavelength is as follows,
Note：In table, fluorescent probe used is all from Molecular Probes,TMInvitrogen
4th step, implements calcium load heart acute over loading intervention, after fluorescent probe loads 5 minutes, uses instead and contain
CaCl2The perfusate heart perfusion of 1.5mmol/L, continues 30 minutes,
5th step, laser confocal microscope is imaged, at 30 μm of centroid adventitia myocardium group of fluorescent collecting flat focus
In knitting, in basic perfusion state, 2 different phases of acute over loading are to heart perfusion, fluorescence signal is continuously scanned, if
Putting sweep interval is 5 minutes, is existed with 514/580,405/580,488/516,633/670 excite/absorbing wavelength respectively
It is imaged under 63 times of oil mirrors, the every frame of scanning speed 0.7 second,
6th step, image procossing and analysis, ImageJ computed in software fluorescence signal intensity.Its Mitochondria O2 -Level is used
Fluorescence intensity ratio under the different absorbing wavelength of 405nm with 514nm for the MitoSox represents, and respectively use MitoGreen and
Mitochondrion representated by Vybrant Ruby and nuclear signal do weight.
Embodiment three
A kind of isolated perfused heart activity chalcogen and the detection method of mitochondrial membrane potential, specially a kind of mice fills in vitro
During stream heart ischemia reperfusion, H in myocardial cell2O2With the real-time in-situ detection method of mitochondrial membrane potential, including following
Step：
The first step, the foundation of mice Isolated Heart perfusion Model, pentobarbital sodium (50mg/kg, i.p.) anesthesia heparin
Change (500IU/kg, i.v.) mice, open rapidly breast and take out heart and be fixed on Langendorff perfusion system, inverse through aorta
Row perfusion is with 95%O2+ 5%CO2The K-H liquid (37 DEG C, pH 7.4, mmol/L) of mixed gas saturation：NaCl 115,
NaHCO325, glucose 11, KCl 4.0, CaCl21.5, MgSO40.9, KH2PO40.9, measure cardiac systolic function for real-time,
Cut an opening in left atrium pulmonary vein porch, a sacculus being connected to pressure transducer is inserted left ventricle through left atrium, complete
It is filled with deionized water until reaching the BDP of 5-10mmHg, by multi-path physiology record system after air in full discharge sacculus
Continuous Real-time Collection left ventricular pressure index, heart rate HR, left ventricular systolic pressure LVSP, left ventricular end diastolic presssure LVEDP, left chamber pressure change
Big speed ± LV dP/dtmaxData, automatically recorded and calculated by computer process and carry out Evaluation on Cardiac Function, treat heart rate and receipts
The perfusate perfused hearts being added with muscle fiber inhibitor Blebbistatin (10mM) are used instead to reduce the heart after contracting function-stable
Rate,
Second step, isolated perfusion system is associated with probe load device and microscopical instrument, using Zeiss LSM 510
Laser Scanning Confocal Microscope, the high optical glass ware of object stage center placement 3cm diameter, 1cm, heart perfusion left ventricular apex portion is affixed on
On glass dish, perfusion pipe end clamp is fixed it is ensured that heart stabilizer is affixed on glass dish avoids displacement, glass dish side wall is put
Put a drainage tube, through peristaltic pump drive can automatic jet heart perfusion effluent, the load of fluorescent probe mainly utilizes micro
Pump installation, the 20X probe concentrated solution being placed in syringe at the uniform velocity pumps into through micro pump, and syringe connects threeway and the heart by flexible pipe
The perfusion terminal of dirty perfusion device is connected, and is adjusted and is controlled fluorescent probe concentrated solution by the opening and closing and micro pump pump speed of threeway
Input opportunity and input speed,
3rd step, fluorescent probe loads, and after isolated heart stablizes 20 minutes under the basic perfusion state, gives for H2O2
Fluorescent probe DCFDA (5X10 with mitochondrial membrane potential-5) and TMRE (5X10 mol/L-6Mol/L), give DAPI (1 μ simultaneously
G/ml) lining dye nucleus, micro pump pumps into 5% that speed is set to heart coronary artery outflow flow velocity, that is,：If mice isolated heart
Coronary outflow flow velocity is 2ml/min, then micro pump pumps into speed and is set to (2ml/min × 5%=0.1ml/min, i.e. 6ml/
H), the art of this patent adopts three carrying methods, used when fluorescent probe concentration used, compound method and fluorescence microscope imaging
Excite/absorbing wavelength is as follows,
Note：In table, fluorescent probe used is all from Molecular Probes,TMInvitrogen
4th step, carries out ischemical reperfusion injury intervention, after fluorescent probe loads 5 minutes, closes perfusion channel, causes
Global ischemia it is seen that whole-heartedly color turn white, myocardial contraction weakens until stop jumping, and perfusion effluent is reduced to zero, and cardioelectric monitor is in
Existing typical case's ischemic change, reopens perfusion channel and forms Reperfu- sion, maintain 60 minutes after 20 minutes,
5th step, laser confocal microscope is imaged, cardiac muscle at about 30 μm of centroid adventitia for the fluorescent collecting flat focus
In tissue, in basic perfusion state, ischemic stage, 3 different phases of Reperfu- sion phase are to heart perfusion, fluorescence signal is carried out continuously
Scanning, setting sweep interval is 5 minutes, respectively with 488/530,543/560,405/458 excite/absorbing wavelength 63
It is imaged under times oil mirror, the every frame of scanning speed 0.7 second,
6th step, image procossing and analysis, ImageJ computed in software fluorescence signal intensity, the fluorescence letter of DCFDA and TMRE
Number do weight with the nuclear signal representated by DAPI respectively.
Claims (4)
1. the detection method of a kind of isolated perfused heart activity chalcogen and mitochondrial membrane potential is it is characterised in that include following walking
Suddenly：The first step, the foundation of Isolated Heart perfusion Model, take small-sized mammalian to open rapidly breast after heparinization, anesthesia and take out the heart
Dirty and be fixed on Langendorff perfusion system, through aorta perfusion retrogradely with 95%O2+ 5%CO2The 37 of mixed gas saturation
The K-H liquid of pH 7.4 at DEG C：The NaHCO of the NaCl of 115mmol/L, 25mmol/L3, the glucose of 11mmol/L, 4.0mmol/
The CaCl of the KCl of L, 1.5mmol/L2, the MgSO of 0.9mmol/L4, the KH of 0.9mmol/L2PO4, for real-time measurement heart contraction
Function, cuts an opening in left atrium pulmonary vein porch, and a sacculus being connected to pressure transducer is inserted the left heart through left atrium
Room, being filled with deionized water until reaching the BDP of 5-10mmHg, being remembered by multi-path physiology after being completely exhausted out air in sacculus
Recording system continuous Real-time Collection left ventricular pressure index, heart rate HR, left ventricular systolic pressure LVSP, left ventricular end diastolic presssure LVEDP, left chamber pressure
Change maximum rate ± LV dP/dtmaxData, automatically recorded and calculated by computer process and carry out Evaluation on Cardiac Function,
Second step, isolated perfusion system is associated with probe load device and microscopical instrument, using Zeiss LSM 510 copolymerization
Focusing microscope, the high optical glass ware of object stage center placement 3-5cm diameter, 1-1.5cm, heart perfusion left ventricular apex portion is pasted
On glass dish, perfusion pipe end clamp is fixed it is ensured that heart stabilizer is affixed on glass dish avoids displacement, glass dish side wall
Place a drainage tube, through peristaltic pump drive can automatic jet heart perfusion effluent, the load of fluorescent probe utilizes micro pump
Device, the probe concentrated solution being placed in syringe at the uniform velocity pumps into through micro pump, and syringe is connect threeway by flexible pipe and filled with heart
The perfusion terminal of stream device is connected, and adjusts, by the opening and closing and micro pump pump speed of threeway, the input controlling fluorescent probe concentrated solution
Opportunity and input speed,
3rd step, fluorescent probe loads, and after isolated heart stablizes 15-20 minute under the basic perfusion state, gives for O2 -、
H2O2, mitochondrion specificity O2 -, and mitochondrial membrane potential fluorescent probe：Using endochylema O2 -During fluorescent probe DHE with 2.5 ×
10-6The Vybrant Ruby lining dye nucleus of mol/L, using endochylema H2O2Served as a contrast with the DAPI of 1 μ g/ml during fluorescent probe DCFDA
Dye nucleus, using mitochondrion specificity O2 -With 2.5 × 10 during fluorescent probe MitoSOX Red-6The Vybrant of mol/L
Ruby and 5 × 10-7The MitoGreen lining dye nucleus of mol/L and mitochondrion, during using mitochondrial membrane potential fluorescent probe TMRE
DAPI and 5 × 10 using 1 μ g/ml-7The MitoGreen lining dye nucleus and mitochondrion of mol/L, fluorescent probe loading concentrations with
Speed pumps into speed controlling by arranging micro pump, and micro pump pumps into speed=probe face liquid concentration/probe concentrated solution concentration *
100%, using bigeminy or three carrying methods, fluorescent probe specificity used, working concentration and fluorescence microscope imaging when institute
With exciting/absorbing wavelength see table,
Note：In table, fluorescent probe used is all from Molecular Probes,TMInvitrogen
4th step, implement Experiment intervention means, include set up isolated heart ischemia-reperfusion, the acute overload of heart, heart tune
Frequency and administration model,
5th step, laser confocal microscope is imaged, fluorescent collecting flat focus in the cardiac muscular tissue at 30 μm of centroid adventitia,
As caused artifact because of heartbeat excessive velocities in image acquisition process, then add in perfusate and use muscle fiber inhibitor 5-10mM
Blebbistatin to reduce heart rate, after fluorescent probe load 10-15 minute, fluorescence signal in heart perfusion is carried out even
Continuous scanning, setting sweep interval is 5 minutes, respectively with exciting corresponding to fluorescent probe used/absorbing wavelength at 63 times
It is imaged under oil mirror, the every frame of scanning speed 0.7 second,
6th step, image procossing and analysis, ImageJ computed in software fluorescence signal intensity, wherein endochylema O2 -Level is existed with DHE
Fluorescence intensity ratio under the different absorbing wavelength of 405nm with 514nm represents, mitochondrion O2 -MitoSox Red is in 405nm for level
Fluorescence intensity ratio under absorbing wavelength different with 514nm represents, endochylema O2 -With the nucleus letter representated by Vybrant Ruby
Number it is weight, endochylema H2O2It is weight, mitochondrion specificity O with the nuclear signal representated by DAPI2 -Use Vybrant Ruby
Do weight with the nucleus representated by MitoGreen and mitochondrial signal, mitochondrial membrane potential fluorescence signal with DAPI and
Nucleus representated by MitoGreen and mitochondrial signal do weight.
2. the detection method of a kind of isolated perfused heart activity chalcogen according to claim 1 and mitochondrial membrane potential, its
It is characterised by, comprise the following steps：
The first step, the foundation of Isolated Heart perfusion Model, rabbit lumbar injection 50mg/kg pentobarbital sodium anaesthetizes intravenous injection simultaneously
500IU/kg heparin, opens rapidly breast and takes out heart be fixed on Langendorff perfusion system, through aorta perfusion retrogradely with
95%O2+ 5%CO2The K-H liquid of pH 7.4 at 37 DEG C of mixed gas saturation：The NaCl of 115mmol/L, 25mmol/L's
NaHCO3, the CaCl of the KCl of the glucose of 11mmol/L, 4.0mmol/L, 1.5mmol/L2, the MgSO of 0.9mmol/L4,
The KH of 0.9mmol/L2PO4, for real-time measurement cardiac systolic function, cut an opening in left atrium pulmonary vein porch, one connected
Sacculus in pressure transducer inserts left ventricle through left atrium, is filled with deionized water until reaching after being completely exhausted out air in sacculus
The BDP of 5-10mmHg, by multi-path physiology record system continuous Real-time Collection left ventricular pressure index, heart rate HR, left room are received
Contractive pressure LVSP, left ventricular end diastolic presssure LVEDP, left chamber pressure change maximum rate ± LV dP/dtmaxData, automatic by computer
Record and calculate process and carry out Evaluation on Cardiac Function, use instead after heart rate and contractile function are stable and be added with muscle fiber inhibitor 5mM
Blebbistatin perfusate perfused hearts to reduce heart rate,
Second step, isolated perfusion system is associated with probe load device and microscopical instrument, using ZeissLSM 510 copolymerization
Focusing microscope, the high optical glass ware of object stage center placement 5cm diameter, 1.5cm, heart perfusion left ventricular apex portion is affixed on glass
On glass ware, perfusion pipe end clamp is fixed it is ensured that heart stabilizer is affixed on glass dish avoids displacement, glass dish side wall is placed
One drainage tube, through peristaltic pump drive can automatic jet heart perfusion effluent, the load of fluorescent probe mainly utilizes micro pump
Device, the 50X probe concentrated solution being placed in syringe at the uniform velocity pumps into through micro pump, and syringe connects threeway and heart by flexible pipe
The perfusion terminal of perfusion device is connected, and is adjusted and is controlled the defeated of fluorescent probe concentrated solution by the opening and closing and micro pump pump speed of threeway
Enter opportunity and input speed,
3rd step, fluorescent probe loads, and after isolated heart stablizes 15 minutes under the basic perfusion state, gives O2 -Fluorescent probe
The concentration of DHE is 5 × 10-6Mol/L, simultaneously with 2.5 × 10-6The Vybrant Ruby lining dye nucleus of mol/L, fluorescent probe
Loading concentrations and speed pump into speed controlling by arranging micro pump, and micro pump pumps into speed and is set to heart coronary artery outflow liquor stream
The 2% of speed, that is,：If rabbit isolated heart coronary outflow flow velocity is 20ml/min, micro pump pumps into speed and is set to 24ml/h,
Computational methods are 20ml/min × 2%=0.4ml/min=24ml/h, and fluorescent probe concentration used, compound method and fluorescence show
Used during micro mirror imaging excite/absorbing wavelength is as follows,
Note：In table, fluorescent probe used is all from Molecular Probes,TMInvitrogen
4th step, implement Experiment intervention means, include set up isolated heart ischemia-reperfusion, the acute overload of heart, heart tune
Frequency and administration model,
5th step, laser confocal microscope is imaged, fluorescent collecting flat focus in the cardiac muscular tissue at 30 μm of centroid adventitia,
As caused artifact because of heartbeat excessive velocities in image acquisition process, then add in perfusate and use muscle fiber inhibitor 5-10mM
Blebbistatin to reduce heart rate, after fluorescent probe load 10 minutes after, fluorescence signal in heart perfusion is carried out continuously
Scanning, fluorescent collecting flat focus in the cardiac muscular tissue at about 30 μm of centroid adventitia, respectively with 514/580,405/580,
633/670 excite/absorbing wavelength is imaged under 63 times of oil mirrors, the every frame of scanning speed 0.7 second,
6th step, image procossing and analysis, ImageJ computed in software fluorescence signal intensity, wherein intracellular O2 -Level is existed with DHE
Fluorescence intensity ratio under the different absorbing wavelength of 405nm with 514nm represents, and is believed with the nucleus representated by Vybrant Ruby
Number do weight.
3. the detection method of a kind of isolated perfused heart activity chalcogen according to claim 1 and mitochondrial membrane potential, its
It is characterised by, comprise the following steps：
The first step, the foundation of isolated rat heart perfusion model, the anesthesia of rats by intraperitoneal injection 50mg/kg pentobarbital sodium is simultaneously quiet
Arteries and veins injects 500IU/kg heparin, opens rapidly breast and takes out heart and be fixed on Langendorff perfusion system, drives in the wrong direction through aorta and fill
Stream is with 95%O2+ 5%CO2The K-H liquid of pH7.4 at 37 DEG C of mixed gas saturation：The NaCl of 115mmol/L, 25mmol/L's
NaHCO3, the CaCl of the KCl of the glucose of 11mmol/L, 4.0mmol/L, 1.5mmol/L2, the MgSO of 0.9mmol/L4,
The KH of 0.9mmol/L2PO4, for real-time measurement cardiac systolic function, cut an opening in left atrium pulmonary vein porch, one connected
Sacculus in pressure transducer inserts left ventricle through left atrium, is filled with deionized water until reaching after being completely exhausted out air in sacculus
The BDP of 5-10mmHg, by multi-path physiology record system continuous Real-time Collection left ventricular pressure index, heart rate HR, left room are received
Contractive pressure LVSP, left ventricular end diastolic presssure LVEDP, left chamber pressure change maximum rate ± LV dP/dtmaxData, automatic by computer
Record and calculate process and carry out Evaluation on Cardiac Function, use instead after heart rate and contractile function are stable and be added with muscle fiber inhibitor 10mM
Blebbistatin perfusate perfused hearts to reduce heart rate,
Second step, isolated perfusion system is associated with probe load device and microscopical instrument, using ZeissLSM 510 copolymerization
Focusing microscope, the high optical glass ware of object stage center placement 3cm diameter, 1cm, heart perfusion left ventricular apex portion is affixed on glass
On ware, perfusion pipe end clamp is fixed it is ensured that heart stabilizer is affixed on glass dish avoids displacement, glass dish side wall places one
Drainage tube, through peristaltic pump drive can automatic jet heart perfusion effluent, the load of fluorescent probe utilizes micro pump installation, will
The 20X probe concentrated solution being placed in syringe at the uniform velocity pumps into through micro pump, and syringe is connect threeway by flexible pipe and filled with cardiac perfusion
The perfusion terminal put is connected, and adjusts, by the opening and closing and micro pump pump speed of threeway, the input opportunity controlling fluorescent probe concentrated solution
And input speed,
3rd step, fluorescent probe loads, and after isolated heart stablizes 15 minutes under the basic perfusion state, gives mitochondrion O2 -Fluorescence
Probe is 2.5 × 10-6The MitoSOX Red of mol/L, simultaneously with 2.5 × 10-6The Vybrant Ruby and 5 × 10 of mol/L- 7The MitoGreen lining dye nucleus of mol/L and mitochondrion, fluorescent probe loading concentrations and speed are pumped into by arranging micro pump
Speed controlling, micro pump pumps into 5% that speed is set to heart coronary artery outflow flow velocity, that is,：If isolated rat heart arteria coronaria stream
Going out flow velocity is 8ml/min, then micro pump pumps into speed and is set to 24ml/h, and computational methods are 8ml/min × 5%=0.4ml/
Min=24ml/h, used when fluorescent probe concentration used, compound method and fluorescence microscope imaging excite/absorbing wavelength is as follows,
Note：In table, fluorescent probe used is all from Molecular Probes,TMInvitrogen
4th step, implements calcium load heart acute over loading intervention, after fluorescent probe loads 5 minutes, uses instead containing CaCl2
The perfusate heart perfusion of 1.5mmol/L, continues 30 minutes,
5th step, laser confocal microscope is imaged, fluorescent collecting flat focus in the cardiac muscular tissue at 30 μm of centroid adventitia,
In basic perfusion state, 2 different phases of acute over loading are to heart perfusion, fluorescence signal is continuously scanned, setting scanning
Interval time is 5 minutes, respectively with 514/580,405/580,488/516,633/670 excite/absorbing wavelength in 63 times of oil
It is imaged under mirror, the every frame of scanning speed 0.7 second,
6th step, image procossing and analysis, ImageJ computed in software fluorescence signal intensity, its Mitochondria O2 -Level is used
Fluorescence intensity ratio under the different absorbing wavelength of 405nm with 514nm for the MitoSox Red represents, and with MitoGreen and
Mitochondrion representated by Vybrant Ruby and nuclear signal do weight.
4. the detection method of a kind of isolated perfused heart activity chalcogen according to claim 1 and mitochondrial membrane potential, its
It is characterised by, comprise the following steps：
The first step, the foundation of mice Isolated Heart perfusion Model, the anesthesia of mouse peritoneal injection 50mg/kg pentobarbital sodium is simultaneously quiet
Arteries and veins injects 500IU/kg heparin, opens rapidly breast and takes out heart and be fixed on Langendorff perfusion system, drives in the wrong direction through aorta and fill
Stream is with 95%O2+ 5%CO2The K-H liquid of pH7.4 at 37 DEG C of mixed gas saturation：The NaCl of 115mmol/L, 25mmol/L's
NaHCO3, the CaCl of the KCl of the glucose of 11mmol/L, 4.0mmol/L, 1.5mmol/L2, the MgSO of 0.9mmol/L4,
The KH of 0.9mmol/L2PO4, for real-time measurement cardiac systolic function, cut an opening in left atrium pulmonary vein porch, one connected
Sacculus in pressure transducer inserts left ventricle through left atrium, is filled with deionized water until reaching after being completely exhausted out air in sacculus
The BDP of 5-10mmHg, by multi-path physiology record system continuous Real-time Collection left ventricular pressure index, heart rate HR, left room are received
Contractive pressure LVSP, left ventricular end diastolic presssure LVEDP, left chamber pressure change maximum rate ± LV dP/dtmaxData, automatic by computer
Record and calculate process and carry out Evaluation on Cardiac Function, use instead after heart rate and contractile function are stable and be added with muscle fiber inhibitor 10mM
Blebbistatin perfusate perfused hearts to reduce heart rate,
Second step, isolated perfusion system is associated with probe load device and microscopical instrument, using Zeiss LSM 510 copolymerization
Focusing microscope, the high optical glass ware of object stage center placement 3cm diameter, 1cm, heart perfusion left ventricular apex portion is affixed on glass
On ware, perfusion pipe end clamp is fixed it is ensured that heart stabilizer is affixed on glass dish avoids displacement, glass dish side wall is placed
One drainage tube, through peristaltic pump drive can automatic jet heart perfusion effluent, the load of fluorescent probe mainly utilizes micro pump
Device, the 20X probe concentrated solution being placed in syringe at the uniform velocity pumps into through micro pump, and syringe connects threeway and heart by flexible pipe
The perfusion terminal of perfusion device is connected, and is adjusted and is controlled the defeated of fluorescent probe concentrated solution by the opening and closing and micro pump pump speed of threeway
Enter opportunity and input speed,
3rd step, fluorescent probe loads, and after isolated heart stablizes 20 minutes under the basic perfusion state, gives for H2O2Glimmering
Light probe is 5 × 10-5The DCFDA of mol/L, gives to be 5 × 10 for the fluorescent probe of mitochondrial membrane potential-6Mol/L's
TMRE, gives the DAPI lining dye nucleus of 1 μ g/ml simultaneously, and micro pump pumps into speed and is set to heart coronary artery outflow flow velocity
5%, that is,：If mice isolated heart coronary outflow flow velocity is 2ml/min, micro pump pumps into speed and is set to 6ml/h, calculates
Method is 2ml/min × 5%=0.1ml/min=6ml/h, using three carrying methods, fluorescent probe concentration used, preparation side
Used when method and fluorescence microscope imaging excite/absorbing wavelength is as follows,
Note：In table, fluorescent probe used is all from Molecular Probes,TMInvitrogen
4th step, carries out ischemical reperfusion injury intervention, after fluorescent probe loads 5 minutes, closes perfusion channel, causes whole-heartedly
Ischemia it is seen that whole-heartedly color turn white, myocardial contraction weakens until stop jumping, and perfusion effluent is reduced to zero, and cardioelectric monitor assumes allusion quotation
Type ischemic change, reopens perfusion channel and forms Reperfu- sion, maintain 60 minutes after 20 minutes,
5th step, laser confocal microscope is imaged, cardiac muscular tissue at about 30 μm of centroid adventitia for the fluorescent collecting flat focus
Interior, in basic perfusion state, ischemic stage, 3 different phases of Reperfu- sion phase are to heart perfusion, fluorescence signal is continuously swept
Retouch, setting sweep interval is 5 minutes, respectively with 488/530,543/560,405/458 excite/absorbing wavelength at 63 times
It is imaged under oil mirror, the every frame of scanning speed 0.7 second,
6th step, image procossing and analysis, ImageJ computed in software fluorescence signal intensity, the fluorescence signal of DCFDA and TMRE divides
Do not do weight with the nuclear signal representated by DAPI.
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