CN103103245A - Method for recording L type calcium channel current by separating guinea pig ventricular muscle cells - Google Patents
Method for recording L type calcium channel current by separating guinea pig ventricular muscle cells Download PDFInfo
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- CN103103245A CN103103245A CN2011103620939A CN201110362093A CN103103245A CN 103103245 A CN103103245 A CN 103103245A CN 2011103620939 A CN2011103620939 A CN 2011103620939A CN 201110362093 A CN201110362093 A CN 201110362093A CN 103103245 A CN103103245 A CN 103103245A
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Abstract
An L type calcium channel is mainly distributed in heart, blood vessels, endocrine and nervous systems, and plays a vitally important role in heart and smooth muscle excitation-contraction coupling, atrionector and atrioventricular node action potential conduction. The method that the influence of a compound on the L type calcium channel is observed through a patch clamp recording method so as to evaluate a hearth safety coefficient of the compound and the effect of treating the hypertension becomes an important research mode. According to the method for recording the L type calcium channel current by separating the guinea pig ventricular muscle cells, through improving a perfusate storage mode, the yield rate and the survival rate of separating the guinea pig ventricular muscle cells are increased, and the stabilization time of the cells is prolonged, and thus the success rate and the stability of experiments are increased.
Description
Technical field
The present invention relates to biotechnology, particularly a kind of separating guinea pig ventricular muscle cell records the method for L-type Calcium Current.
Background technology
The L-type calcium channel mainly is distributed in cardiac muscle, unstriated muscle (comprising blood vessel, intestines, lung, uterus), endocrine cell (comprising pancreatic β cell, hypophysis) and nerve.The L-type calcium channel plays an important role in conduction, synaptic plasticity and the hormone secretion of the action potential of E-C coupling, sinus node and the atrioventricular node of heart and unstriated muscle.
In the performance history of hypertension, cardiovascular disorder and cancer drug, the antagonistic action of observing the L-type calcium channel has become the important means of assisting to estimate drug effect.Patch clamp technique is called as " gold standard " of research ionic channel, is the most important technology of research ionic channel.The ventricular muscle cell of isolating cardiac records the L-type calcium channel with full cell patch pincers mode has become important step in drug discovery process in order to the impact of estimating medicine.
The ways and means that conventional myocardial cell separates comprises the digestion of collagenase and the process of multiple calcium.The defectives such as there is complicated operation in this traditional separation method, and cell harvesting rate and surviving rate are low; Directly cancelled Na in the perfusate of extracellular
+, cause the obvious defective such as shortening of cell survival time, need to improve and improve, thereby guarantee success ratio and the stability of testing.
Summary of the invention
Purpose of the present invention, be to overcome the defective that above-mentioned prior art exists, provide a kind of easy to operate, method that separating guinea pig ventricular muscle cell that can improve cell harvesting rate and surviving rate records the L-type Calcium Current, with safety evaluation that a kind of clinical front heart is provided and the effective ways of hypertension cancer therapy drug evaluating drug effect.
The object of the present invention is achieved like this: a kind of separating guinea pig ventricular muscle cell records the method for L-type Calcium Current, comprises the following steps:
A, cavy is fiercelyed attack after head causes stupor, opened chest and take out fast heart;
B, guinea pig heart is placed in the normal Tai Shi solution of 4 ℃ removes fat and pericardium, separate aorta and intubate, be placed in and keep 37 ℃ of speed with 8-10ml/min of temperature to carry out the isolated heart perfusion on the Langendorff device: first with normal Tai Shi solution perfusion 5min, rinse residual blood in heart fully, use again without calcium Tai Shi solution perfusion 5min, stop fully beating to heart, use again 50ml enzymolysis solution circulation perfusion 8-10min, obvious to the wire drawing of heart effluent liquid, expand and become large, when color is thin out, then use Kraft-Br ü he solution perfusion 5min;
C, heart is taken off from the Langendorff device, cuts off atrium and basis pontis tissue, the ventricular muscles tissue is cut into the 1.5-2.5mm fragment, with thick opening suction pipe slowly piping and druming ventricular muscle cell is separated from myocardium fragment;
D, isolated ventricular muscle cell is placed in 37 ℃ of water-baths 1-2min that vibrates, obtains cell suspension;
E, with cell suspension with 200 purpose strainer filterings, ventricular muscle cell is collected in Kraft-Br ü he solution, and is standby after standing stable 0.5-1h under room temperature;
F, the ventricular muscle cell that will be collected in Kraft-Br ü he solution add in the perfusion groove, are placed on the inverted microscope Stage microscope, and standing rear ventricular muscle cell sinks to the bottom adherent, clean cell with the extracellular perfusate, and flow velocity keeps 1-1.5ml/min; Select neat in edge, the surface is without particle, and band is clear, and shrinkage-free ventricular muscle cell carries out patch clamp experiments, adopts the full cell patch pincers recording method of standard, records the L-type Calcium Current under the voltage clamp pattern; Experiment is carried out under 22-25 ℃, and the resistance of the glass electrode of using when being full of intracellular perfusion liquid is 3-5M Ω.
Normal Tai Shi solution described in step B is by NaCl, KCl, MgCl
2, CaCl
2, 4-hydroxyethyl piperazine ethanesulfonic acid, glucose be formulated, and regulates pH to 7.4 with NaOH, in the solution that is mixed with, the concentration of NaCl is 135mM, the concentration of KCl is 5.4mM, MgCl
2Concentration be 1mM, CaCl
2Concentration be 1.8mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
Described without calcium Tai Shi solution by NaCl, KCl, MgCl
2, 4-hydroxyethyl piperazine ethanesulfonic acid, glucose be formulated, and regulates pH to 7.4 with NaOH, in the solution that is mixed with, the concentration of NaCl is 135mM, the concentration of KCl is 5.4mM, MgCl
2Concentration be 1mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
Described Kraft-Br ü he solution is by taurine, oxalic acid, L-glutamic acid, KCl, KH
2PO
4, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 4-hydroxyethyl piperazine ethanesulfonic acid, glucose be formulated, and regulates pH to 7.4 with KOH, in the solution that is mixed with, the concentration of taurine is 10mM, and the concentration of oxalic acid is 10mM, and the concentration of L-glutamic acid is 70mM, the concentration of KCl is 25mM, KH
2PO
4Concentration be 10mM, the concentration of ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) is 0.5mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 5mM, the concentration of glucose is 11mM; The osmotic pressure of solution is 290mOsm.
Extracellular perfusate described in step F is by NaCl, CsCl, MgCl
2, CaCl
2, 4-hydroxyethyl piperazine ethanesulfonic acid, glucose be formulated, and regulates pH to 7.4 with NaOH, in the solution that is mixed with, the concentration of NaCl is 135mM, the concentration of CsCl is 5.4mM, MgCl
2Concentration be 1mM, CaCl
2Concentration be 1.8mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
Described intracellular perfusion liquid is by CsCl, etamon chloride, CaCl
2, magnesium-Triphosaden, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 4-hydroxyethyl piperazine ethanesulfonic acid be formulated, and regulates pH to 7.2 with CsOH, in the solution that is mixed with, the concentration of CsCl is 120mM, the concentration of etamon chloride is 20mM, CaCl
2Concentration be 1mM, the concentration of magnesium-Triphosaden is 5mM, the concentration of ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) is 11mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 10mM; The osmotic pressure of solution is 310mOsm.
Voltage clamp pattern described in step F is, clamps down on voltage-80mV, first bestow 50ms-40mV prepulsing, after give 300ms, 0mV depolarize pulse.
After all passing to 100% oxygen 15_min, uses each perfusion solution described in step B.
Under room temperature described in step e, be 30_min standing steady time.
The advantage that separating guinea pig ventricular muscle cell of the present invention records the method for L-type Calcium Current is:
1, the abundant residual collagenase in cleansing tissue of the perfusion of Kraft-Br ü he solution, avoided the impact of removing residual glue protoenzyme on cell, improved yield rate and the surviving rate of cell;
2, the ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) in Kraft-Br ü he solution enters the calcium ion that dissociates in abundant chelating born of the same parents in cell, weakens myocardial cell's Spontaneous Contraction, has improved the surviving rate of cell;
But the survival time and the good order and condition that 3, have added the nutrition significant prolongation cells such as taurine, L-glutamic acid in Kraft-Br ü he liquid;
4, normally added Na in the perfusate of extracellular
+, kept the balance of the inside and outside ion of cell, the stability that effectively extended cell;
5, the separating obtained pig ventricular myocytes of the present invention has normal ion channel function, is applicable to patch clamp technique to the research of cardiac electrophysiological characteristic, and this experimental implementation is easy to grasp, and is a kind of method of simple and effective acute isolation ventricular muscle cell.
Embodiment
Separating guinea pig ventricular muscle cell of the present invention records the method for L-type Calcium Current, comprises the following steps:
One, the separation of Ventricular Myocytes
Cavy (body weight 220-250g) is fiercelyed attack after head causes stupor, opens chest and takes out fast heart, removes fat and pericardium in 4 ℃ of normal tyrode's solutions, separates aorta and intubate, carries out the isolated heart perfusion with the speed of 8-10ml/min.First rinse blood residual in heart fully with normal Zinciodati Comp solution perfusion 5min, use again without calcium Zinciodati Comp solution perfusion 5min and stop beating fully to heart, use again 50ml enzymolysis solution circulation perfusion 8-10min, obvious to the wire drawing of heart effluent liquid, heart expands and becomes large, when color is thin out, after Kraft-Br ü he liquid perfusion 5min, heart is taken off from the Langendorff device, cut off atrium and basis pontis tissue, with the ventricular muscles tissue shear into about the 2mm fragment, with thick opening suction pipe slowly piping and druming cell is separated from myocardium fragment, be placed in 37 ℃ of water-baths 1-2min that vibrates.Cell suspension is collected in Kraft-Br ü he liquid with 200 purpose strainer filterings, myocardial cell, under room temperature after standing stable 0.5-1h (preferably 0.5h) standby.Whole perfusion system temperature remains on 37 ℃, flow velocity 8-10ml/min.All perfusates use after all passing to 100% oxygen 15min.
Two, the record of L-type Calcium Current
Cell is added in the perfusion groove, be placed on the inverted microscope Stage microscope, sink to the bottom adherently after cell is standing, clean cell with extracellular perfusate perfusion, flow velocity keeps 1-1.5ml/min.Select neat in edge, the surface is without particle, and band is clear, and shrinkage-free cell carries out patch clamp experiments.All experiments are carried out under room temperature (22-25 ℃).The resistance of glass electrode when being full of intracellular perfusion liquid that uses is about 3-5M Ω.The full cell patch pincers recording method of employing standard, recording channel electric current under the voltage clamp pattern.When recording the L-type Calcium Current, clamp down on voltage-80mV, first bestow 50ms-40mV prepulsing, after give 300ms, 0mV depolarize pulse.
The normal Tai Shi solution that adopts in the present invention is by NaCl, KCl, MgCl
2, CaCl
2, 4-hydroxyethyl piperazine ethanesulfonic acid, glucose be formulated, and regulates pH to 7.4 with NaOH, in the solution that is mixed with, the concentration of NaCl is 135mM, the concentration of KCl is 5.4mM, MgCl
2Concentration be 1mM, CaCl
2Concentration be 1.8_mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
Adopt in the present invention without calcium Tai Shi solution by NaCl, KCl, MgCl
2, 4-hydroxyethyl piperazine ethanesulfonic acid, glucose be formulated, and regulates pH to 7.4 with NaOH, in the solution that is mixed with, the concentration of NaCl is 135mM, the concentration of KCl is 5.4mM, MgCl
2Concentration be 1mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
The Kraft-Br ü he solution that adopts in the present invention is by taurine, oxalic acid, L-glutamic acid, KCl, KH
2PO
4, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 4-hydroxyethyl piperazine ethanesulfonic acid, glucose be formulated, and regulates pH to 7.4 with KOH, in the solution that is mixed with, the concentration of taurine is 10mM, and the concentration of oxalic acid is 10mM, and the concentration of L-glutamic acid is 70mM, the concentration of KCl is 25mM, KH
2PO
4Concentration be 10mM, the concentration of ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) is 0.5mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 5mM, the concentration of glucose is 11mM; The osmotic pressure of solution is 290mOsm.
The extracellular perfusate that adopts in the present invention is by NaCl, CsCl, MgCl
2, CaCl
2, 4-hydroxyethyl piperazine ethanesulfonic acid, glucose be formulated, and regulates pH to 7.4 with NaOH, in the solution that is mixed with, the concentration of NaCl is 135mM, the concentration of CsCl is 5.4mM, MgCl
2Concentration be 1mM, CaCl
2Concentration be 1.8mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
The intracellular perfusion liquid that adopts in the present invention is by CsCl, etamon chloride, CaCl
2, magnesium-Triphosaden, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 4-hydroxyethyl piperazine ethanesulfonic acid be formulated, and regulates pH to 702 with CsOH, in the solution that is mixed with, the concentration of CsCl is 120mM, the concentration of etamon chloride is 20mM, CaCl
2Concentration be 1mM, the concentration of magnesium-Triphosaden is 5mM, the concentration of ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) is 11mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 10mM; The osmotic pressure of solution is 310mOsm.
Claims (6)
1. a separating guinea pig ventricular muscle cell records the method for L-type Calcium Current, it is characterized in that, comprises the following steps:
A, cavy is fiercelyed attack after head causes stupor, opened chest and take out fast heart;
B, guinea pig heart is placed in the normal Tai Shi solution of 4 ℃ removes fat and pericardium, separate aorta and intubate, be placed in and keep 37 ℃ of speed with 8-10ml/min of temperature to carry out the isolated heart perfusion on the Langendorff device: first with normal Tai Shi solution perfusion 5min, rinse residual blood in heart fully, use again without calcium Tai Shi solution perfusion 5min, stop fully beating to heart, use again 50ml enzymolysis solution circulation perfusion 8-10min, obvious to the wire drawing of heart effluent liquid, expand and become large, when color is thin out, then use Kraft-Br ü he solution perfusion 5min;
C, heart is taken off from the Langendorff device, cuts off atrium and basis pontis tissue, the ventricular muscles tissue is cut into the 1.5-2.5mm fragment, with thick opening suction pipe slowly piping and druming ventricular muscle cell is separated from myocardium fragment;
D, isolated ventricular muscle cell is placed in 37 ℃ of water-baths 1-2min that vibrates, obtains cell suspension;
E, with cell suspension with 200 purpose strainer filterings, ventricular muscle cell is collected in Kraft-Br ü he solution, and is standby after standing stable 0.5-1h under room temperature;
F, the ventricular muscle cell that will be collected in Kraft-Br ü he solution add in the perfusion groove, are placed on the inverted microscope Stage microscope, and standing rear ventricular muscle cell sinks to the bottom adherent, clean cell with the extracellular perfusate, and flow velocity keeps 1-1.5ml/min; Select neat in edge, the surface is without particle, and band is clear, and shrinkage-free ventricular muscle cell carries out patch clamp experiments, adopts the full cell patch pincers recording method of standard, records the L-type Calcium Current under the voltage clamp pattern; Experiment is carried out under 22-25 ℃, and the glass electrode of use is full of intracellular perfusion liquid, and resistance is 3-5M Ω.
2. separating guinea pig ventricular muscle cell according to claim 1 records the method for L-type Calcium Current, it is characterized in that: the normal Tai Shi solution described in step B is by NaCl, KCl, MgCl
2, CaCl
2, 4-hydroxyethyl piperazine ethanesulfonic acid, glucose be formulated, and regulates pH to 7.4 with NaOH, in the solution that is mixed with, the concentration of NaCl is 135mM, the concentration of KCl is 5.4mM, MgCl
2Concentration be 1mM, CaCl
2Concentration be 1.8_mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
Described without calcium Tai Shi solution by NaCl, KCl, MgCl
2, 4-hydroxyethyl piperazine ethanesulfonic acid, glucose be formulated, and regulates pH to 7.4 with NaOH, in the solution that is mixed with, the concentration of NaCl is 135mM, the concentration of KCl is 5.4mM, MgCl
2Concentration be 1mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
Described Kraft-Br ü he solution is by taurine, oxalic acid, L-glutamic acid, KCl, KH
2PO
4, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 4-hydroxyethyl piperazine ethanesulfonic acid, glucose be formulated, and regulates pH to 7.4 with KOH, in the solution that is mixed with, the concentration of taurine is 10mM, and the concentration of oxalic acid is 10mM, and the concentration of L-glutamic acid is 70mM, the concentration of KCl is 25mM, KH
2PO
4Concentration be 10mM, the concentration of ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) is 0.5mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 5mM, the concentration of glucose is 11mM; The osmotic pressure of solution is 290mOsm.
3. separating guinea pig ventricular muscle cell according to claim 1 records the method for L-type Calcium Current, it is characterized in that: the extracellular perfusate described in step F is by NaCl, CsCl, MgCl
2, CaCl
2, 4-hydroxyethyl piperazine ethanesulfonic acid, glucose be formulated, and regulates pH to 7.4 with NaOH, in the solution that is mixed with, the concentration of NaCl is 135mM, the concentration of CsCl is 5.4mM, the concentration of MgCl2 is 1mM, CaCl
2Concentration be 1.8mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
Described intracellular perfusion liquid is by CsCl, etamon chloride, CaCl
2, magnesium-Triphosaden, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 4-hydroxyethyl piperazine ethanesulfonic acid be formulated, and regulates pH to 7.2 with CsOH, in the solution that is mixed with, the concentration of CsCl is 120mM, the concentration of etamon chloride is 20mM, CaCl
2Concentration be 1mM, the concentration of magnesium-Triphosaden is 5mM, the concentration of ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) is 11mM, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 10mM; The osmotic pressure of solution is 310mOSm.
4. separating guinea pig ventricular muscle cell according to claim 1 records the method for L-type Calcium Current, it is characterized in that: the voltage clamp pattern described in step F is, clamp down on voltage-80mV, first bestow 50ms-40mV prepulsing, after give 300ms, 0mV depolarize pulse.
5. separating guinea pig ventricular muscle cell according to claim 1 records the method for L-type Calcium Current, it is characterized in that: use after each perfusion solution described in step B all passes to 100% oxygen 15min.
6. separating guinea pig ventricular muscle cell according to claim 1 records the method for L-type Calcium Current, it is characterized in that: under the room temperature described in step e, be 30min standing steady time.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877959A (en) * | 2015-05-11 | 2015-09-02 | 南通科瑞斯生物医药科技有限公司 | Culture method for isolated cardiomyocytes of adult guinea pig |
| CN105527462A (en) * | 2016-01-21 | 2016-04-27 | 长春理工大学 | Method for measuring single alive myocardial cell action potential and pulsing force by atomic force microscope |
-
2011
- 2011-11-15 CN CN2011103620939A patent/CN103103245A/en active Pending
Non-Patent Citations (7)
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| ANDREW F. JAMES: "Gender-related differences in ventricular myocyte repolarization in the guinea pig", 《BASIC RES CARDIOL》 * |
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| 刘妍妍等: "白藜芦醇对豚鼠心室肌细胞L型钙通道的影响", 《中国药理学通报》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877959A (en) * | 2015-05-11 | 2015-09-02 | 南通科瑞斯生物医药科技有限公司 | Culture method for isolated cardiomyocytes of adult guinea pig |
| CN105527462A (en) * | 2016-01-21 | 2016-04-27 | 长春理工大学 | Method for measuring single alive myocardial cell action potential and pulsing force by atomic force microscope |
| CN105527462B (en) * | 2016-01-21 | 2018-01-02 | 长春理工大学 | A kind of method that AFM measures single Single Cardiac Cell living and pulsating force |
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Application publication date: 20130515 |