CN102809595A - Method for recording Nav 1.5 sodium channel current by separating cavy ventricular muscle cells - Google Patents
Method for recording Nav 1.5 sodium channel current by separating cavy ventricular muscle cells Download PDFInfo
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- CN102809595A CN102809595A CN201210299397XA CN201210299397A CN102809595A CN 102809595 A CN102809595 A CN 102809595A CN 201210299397X A CN201210299397X A CN 201210299397XA CN 201210299397 A CN201210299397 A CN 201210299397A CN 102809595 A CN102809595 A CN 102809595A
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Abstract
The invention provides a method for recording an Nav 1.5 sodium channel current by separating cavy ventricular muscle cells. The method comprises two steps of separating a single cavy ventricular muscle cell and recording the Nav 1.5 sodium channel current. According to the method, the yield rate and the survival rate of the separated cavy ventricular muscle cells are enhanced by improvement of a perfusate and a storage way, and the success rate and the stability of an experiment are increased by the prolonging of the stable time of the cell. The separated cavy ventricular muscle cell has a normal ion channel function and the method is suitable to research on a ventricular muscle physiological characteristic by a patch clamp technique; the experiment operation is easy to master, and the method for acutely separating the ventricular muscle cells is simple and efficient.
Description
Technical field
The present invention relates to biotechnology, particularly a kind of method of separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current.
Background technology
The Nav1.5 sodium channel is main myocardium voltage-gated sodium channel, the sodium channel of expressing one's gratification again.The density of sodium-ion channel is maximum on human myocardium's cell, and the sodium-ion channel that high density distributes is to the formation of normal impulsion and propagate significant.INav1.5 density and dynamic (dynamical) change can be disturbed conduction and prolong multipole.
Research shows, the LQT syndrome, and the Brugada syndrome, (progressive cardiacconduetion defect, PECD), multiple heart diseases such as diseased sinus node syndrome are relevant with the heart sodium-ion channel for carrying out property cardiac conduction obstacle.
In the performance history of angiocardiopathy, the antagonism of observing the Nav1.5 sodium channel has become the important means of assisting to estimate drug effect.Patch clamp technique is called as " goldstandard " of research ion channel, is the most important technology of research ion channel.The ventricular muscle cell of isolating cardiac writes down the Nav1.5 sodium channel with full cell patch pincers mode has become the important step in the drug discovery process in order to the influence of estimating medicine.
The ways and means that conventional cardiac muscle cell separates comprises the digestion of clostridiopetidase A and the process of multiple calcium.Defectives such as there is complicated operation in this traditional separation method, and cell harvesting rate and survival rate are low; Directly cancel calcium ion in the perfusate of extracellular, caused obvious defective such as shortening of cell survival time, need improve and improve, thereby guaranteed the success ratio and the stability of testing.
Summary of the invention
The object of the invention is to overcome the defective that above-mentioned prior art exists, and a kind of method of easy to operate, the separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current that can improve cell harvesting rate and survival rate is provided.
The objective of the invention is to realize like this: a kind of method of separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current may further comprise the steps:
A, cavy is fiercelyed attack after head causes stupor, opened chest and take out heart fast;
B, place 4 ℃ normal Tai Shi solution to remove fat and pericardium guinea pig heart, separate sustainer and intubate, place to keep 37 ℃ of speed of temperature to carry out the isolated heart perfusion on the Langendorff device: earlier with normal Tai Shi solution perfusion 5min with 6-8ml/min; Residual blood in the complete flushing heart; With no calcium Tai Shi solution perfusion 5min, stop fully beating again, use 50ml enzymolysis liquid circulation perfusion 8-10min again to heart; Obvious to the wire drawing of heart effluent; Expand to become big, when color is thin out, use Kraft-Br ü he solution perfusion 5min (perfusion of 5 minutes Kraft-Br ü he liquid fully cleansing tissue in residual clostridiopetidase A) again;
C, heart is taken off from the Langendorff device, cuts off atrium and basal part 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 37 ℃ of water-baths 1-2min that vibrates, obtain cell suspension;
E, with cell suspension with 200 purpose strainer filterings, ventricular muscle cell is collected in the Kraft-Br ü he solution, leave standstill under the room temperature stablize behind the 0.5-1h subsequent use;
F, the ventricular muscle cell that will be collected in the Kraft-Br ü he solution add in the perfusion groove, place on the inverted microscope objective table, leave standstill the back ventricular muscle cell and sink to the bottom adherently, clean cell with the extracellular perfusate, flow velocity maintenance 1-1.5ml/min; Select neat in edge, the no particle in surface, band is clear, and shrinkage-free ventricular muscle cell carries out patch clamp experiments, adopts the full cell patch pincers recording method of standard, record Nav1.5 sodium channel 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.
The method of above-mentioned separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current, wherein, the normal Tai Shi solution described in the step B is by NaCl, KCl, MgCl
2, CaCl
2, 4-HEPES, 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-HEPES is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
The method of above-mentioned separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current, wherein, no calcium Tai Shi solution is by NaCl, KCl, MgCl
2, 4-HEPES, 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-HEPES is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
The method of above-mentioned separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current, wherein, Kraft-Br ü he solution is by tarine, ethane diacid, glutamic acid, KCl, KH
2PO
4, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 4-HEPES, glucose be formulated, and regulates pH to 7.4 with KOH, in the solution that is mixed with; The concentration of tarine is 10mM, and the concentration of ethane diacid is 10mM, and the concentration of 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-HEPES is 5mM, the concentration of glucose is 11mM; The osmotic pressure of solution is 290mOsm.Ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) in the Kraft-Br ü he solution can get into and fully sting the calcium ion that closes endocellular liberation in the cell; Avoided cardiac muscle cell's spontaneity to shrink; But the holding time of significant prolongation cell, and do not influence the normal electrophysiological characteristics of cell.
The method of above-mentioned separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current, wherein, the extracellular perfusate described in the step F is by NaCl, CsCl, MgCl
2, CaCl
2, 4-HEPES, glucose, CdCl
2Formulated, and with NaOH adjusting pH to 7.4, 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-HEPES is 5mM, the concentration of glucose is 10mM, CdCl
2Concentration be 0.5mM; The osmotic pressure of solution is 290mOsm;
The method of above-mentioned separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current, wherein, intracellular perfusion liquid is by CsCl, etamon chloride, CaCl
2, magnesium-atriphos, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 4-HEPES 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-atriphos is 5mM, the concentration of ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) is 11mM, the concentration of 4-HEPES is 10mM; The osmotic pressure of solution is 310mOSm.
The method of above-mentioned separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current, wherein, the voltage clamp pattern described in the step F does, clamps down on voltage-110mV, bestow 20ms-30mV pulse command voltage.
The method of above-mentioned separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current wherein, is used after each the perfusion solution described in the step B all passes to 100% oxygen 15min.
The method of above-mentioned separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current, wherein, leaving standstill stabilization time under the room temperature described in the step e is 30min.
The advantage of the method for separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current of the present invention is:
1, the abundant residual clostridiopetidase A in the cleansing tissue of the perfusion of Kraft-Br ü he liquid has been avoided the influence of residual clostridiopetidase A pair cell, has improved the yield rate and the survival rate of cell;
2, the ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) in the Kraft-Br ü he liquid gets into the calcium ion that dissociates in the abundant chelating born of the same parents in the cell, weakens cardiac muscle cell's spontaneity and shrinks, and has improved the survival rate of cell;
3, added tarine in the Kraft-Br ü he liquid, but the time-to-live and the kilter of nutrients significant prolongation cells such as glutamic acid;
4, normally add sodium, caesium, calcium and magnesium ion in the perfusate of extracellular, kept the balance of the inside and outside ion of cell, the stability that has effectively prolonged cell.
In a word, the separating obtained myocyte of guinea-pig ventricular of the present invention has normal ion channel function, is applicable to patch clamp technique to the cardiac electrophysiology The Characteristic Study, and this experimental implementation is easy to grasp, and is a kind of method of simple and effective acute isolation ventricular muscle cell.
Embodiment
The method of separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current of the present invention may further comprise the steps:
One, the separation of single ventricular muscle cell
After cavy (the body weight 220-250 gram) head of fiercelying attack causes stupor, open chest and take out heart fast, in 4 degrees centigrade of normal tyrode's solutions, remove fat and pericardium, separate sustainer and intubate, the speed of 6-8 ml/min is carried out the Langendorff cardiac perfusion.Earlier with residual blood in 5 minutes complete flushing hearts of normal normal Zinciodati Comp solution perfusion, then stop fully beating to heart with no calcium Zinciodati Comp solution perfusion 5 minutes, use 50 milliliters of enzymolysis liquid circulation perfusions 8-10 minute again; Obvious to the wire drawing of heart effluent, heart expands and becomes big, when color is thin out; With Kraft-Br ü he liquid perfusion after 5 minutes; Heart is taken off from the Langendorff device, cut off atrium and basal part tissue, the ventricular muscles tissue is cut into about 2 millimeters fragments; With the slow piping and druming of thick opening suction pipe cell is separated from myocardium fragment, place 37 degrees centigrade of water-baths vibrations 1-2 minute.Cell suspension is with 200 purpose strainer filterings, and the cardiac muscle cell is collected in the Kraft-Br ü he liquid, leave standstill under the room temperature stablize after 1 hour subsequent use.Whole perfusion system temperature remains on 37 degrees centigrade, flow velocity 8-10 ml/min.All perfusates all pass to 100% oxygen and use after 15 minutes.
Two, the record of Nav1.5 sodium channel current
Cell is added in the perfusion groove, place on the inverted microscope objective table, sink to the bottom adherently after cell leaves standstill, extracellular perfusate perfusion cleans cell, and flow velocity keeps the 1-1.5 ml/min.Select neat in edge, the no particle in surface, band is clear, and shrinkage-free cell carries out patch clamp experiments.All experiments are carried out under room temperature (22-25 degree centigrade).The resistance of glass electrode when being full of intracellular perfusion liquid that uses is about the 3-5 megaohm.The full cell patch pincers recording method of employing standard, recording channel electric current under the voltage clamp pattern.During record Nav1.5 sodium channel current, clamp down on voltage-110 millivolt, bestow 20 milliseconds-30 millivolts command voltage.
The normal Tai Shi solution that is adopted among the present invention is by NaCl, KCl, MgCl
2, CaCl
2, 4-HEPES, 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-HEPES is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
The no calcium Tai Shi solution that is adopted among the present invention is by NaCl, KCl, MgCl
2, 4-HEPES, 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-HEPES is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
The Kraft-Br ü he solution that is adopted among the present invention is by tarine, ethane diacid, glutamic acid, KCl, KH
2PO
4, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 4-HEPES, glucose be formulated, and regulates pH to 7.4 with KOH, in the solution that is mixed with; The concentration of tarine is 10mM, and the concentration of ethane diacid is 10mM, and the concentration of 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-HEPES is 5mM, the concentration of glucose is 11mM; The osmotic pressure of solution is 290mOsm.
The extracellular perfusate that is adopted among the present invention is by NaCl, CsCl, MgCl
2, CaCl
2, 4-HEPES, glucose, CdCl
2Formulated, and with NaOH adjusting pH to 7.4, 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-HEPES is 5mM, the concentration of glucose is 10mM, CdCl
2Concentration be 0.5mM; The osmotic pressure of solution is 290mOsm;
The intracellular perfusion liquid that is adopted among the present invention is by CsCl, etamon chloride, CaCl
2, magnesium-atriphos, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 4-HEPES 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-atriphos is 5mM, the concentration of ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) is 11mM, the concentration of 4-HEPES is 10mM; The osmotic pressure of solution is 310mOSm.
Claims (6)
1. the method for a separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current is characterized in that, may further comprise the steps:
A, cavy is fiercelyed attack after head causes stupor, opened chest and take out heart fast;
B, place 4 ℃ normal Tai Shi solution to remove fat and pericardium guinea pig heart, separate sustainer and intubate, place to keep 37 ℃ of speed of temperature to carry out the isolated heart perfusion on the Langendorff device: earlier with normal Tai Shi solution perfusion 5min with 6-8ml/min; Residual blood in the complete flushing heart; With no calcium Tai Shi solution perfusion 5min, stop fully beating again, use 50ml enzymolysis liquid circulation perfusion 8-10min again to heart; Obvious to the wire drawing of heart effluent; Expanding becomes big, when color is thin out, uses Kraft-Br ü he solution perfusion 5min again;
C, heart is taken off from the Langendorff device, cuts off atrium and basal part 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 37 ℃ of water-baths 1-2min that vibrates, obtain cell suspension;
E, with cell suspension with 200 purpose strainer filterings, ventricular muscle cell is collected in the Kraft-Br ü he solution, leave standstill under the room temperature stablize behind the 0.5-1h subsequent use;
F, the ventricular muscle cell that will be collected in the Kraft-Br ü he solution add in the perfusion groove, place on the inverted microscope objective table, leave standstill the back ventricular muscle cell and sink to the bottom adherently, clean cell with the extracellular perfusate, flow velocity maintenance 1-1.5ml/min; Select neat in edge, the no particle in surface, band is clear, and shrinkage-free ventricular muscle cell carries out patch clamp experiments, adopts the full cell patch pincers recording method of standard, record Nav1.5 sodium channel 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. the method for separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current according to claim 1, it is characterized in that: the normal Tai Shi solution described in the step B is by NaCl, KCl, MgCl
2, CaCl
2, 4-HEPES, 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-HEPES is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
Described no calcium Tai Shi solution is by NaCl, KCl, MgCl
2, 4-HEPES, 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-HEPES is 5mM, the concentration of glucose is 10mM; The osmotic pressure of solution is 290mOsm;
Described Kraft-Br ü he solution is by tarine, ethane diacid, glutamic acid, KCl, KH
2PO
4, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 4-HEPES, glucose be formulated, and regulates pH to 7.4 with KOH, in the solution that is mixed with; The concentration of tarine is 10mM, and the concentration of ethane diacid is 10mM, and the concentration of 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-HEPES is 5mM, the concentration of glucose is 11mM; The osmotic pressure of solution is 290mOsm.
3. the method for separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current according to claim 1, it is characterized in that: the extracellular perfusate described in the step F is by NaCl, CsCl, MgCl
2, CaCl
2, 4-HEPES, glucose, CdCl
2Formulated, and with NaOH adjusting pH to 7.4, 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-HEPES is 5mM, the concentration of glucose is 10mM, CdCl
2Concentration be 0.5mM; The osmotic pressure of solution is 290mOsm;
Described intracellular perfusion liquid is by CsCl, etamon chloride, CaCl
2, magnesium-atriphos, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), 4-HEPES 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-atriphos is 5mM, the concentration of ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) is 11mM, the concentration of 4-HEPES is 10mM; The osmotic pressure of solution is 310mOSm.
4. the method for separating guinea pig ventricular muscle cell according to claim 1 record Nav1.5 sodium channel current is characterized in that: the voltage clamp pattern described in the step F does, clamps down on voltage-110mV, bestow 20ms-30mV pulse command voltage.
5. the method for separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current according to claim 1 is characterized in that: use after each the perfusion solution described in the step B all passes to 100% oxygen 15min.
6. the method for separating guinea pig ventricular muscle cell record Nav1.5 sodium channel current according to claim 1, it is characterized in that: leaving standstill stabilization time under the room temperature described in the step e is 30min.
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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 |
| CN105675855A (en) * | 2016-01-21 | 2016-06-15 | 天津工业大学 | Low-frequency magnetic field generator for cell experiment researches |
| CN108004205A (en) * | 2017-12-14 | 2018-05-08 | 华中科技大学同济医学院附属协和医院 | Myocardium buffer kit and application method for cardiac muscle cell's separating-purifying |
-
2012
- 2012-08-21 CN CN201210299397XA patent/CN102809595A/en active Pending
Non-Patent Citations (2)
| Title |
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| 史钰芳 等: "《抗Nav1.5抗体对豚鼠心室肌细胞钠电流的影响》", 《中华急诊医学杂志》 * |
| 王国涛: "《雷诺嗪对豚鼠心率失常及心功能影响的实验研究》", 《中国博士学位论文全文数据库》 * |
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 |
| CN105675855A (en) * | 2016-01-21 | 2016-06-15 | 天津工业大学 | Low-frequency magnetic field generator for cell experiment researches |
| CN108004205A (en) * | 2017-12-14 | 2018-05-08 | 华中科技大学同济医学院附属协和医院 | Myocardium buffer kit and application method for cardiac muscle cell's separating-purifying |
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Application publication date: 20121205 |