CN102488923B - Tissue-engineered atrioventricular node and construction method and application thereof - Google Patents
Tissue-engineered atrioventricular node and construction method and application thereof Download PDFInfo
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- CN102488923B CN102488923B CN201110415250.8A CN201110415250A CN102488923B CN 102488923 B CN102488923 B CN 102488923B CN 201110415250 A CN201110415250 A CN 201110415250A CN 102488923 B CN102488923 B CN 102488923B
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Abstract
The invention relates to the technical field of biomedical engineering. At present, the research about the construction of a tissue-engineered atrioventricular node is not reported in China and foreign countries. The invention provides a method for constructing the tissue-engineered atrioventricular node by using cardiac pacing cells and conduction cells which are obtained by inducing collagen sponge and bone marrow mesenchymal stem cells. The bone marrow mesenchymal stem cells are induced into the pacing cells through co-culture with sinoatrial node cells, and are induced into the cardiac pacing cells by a method for co-culture with auricular appendage muscle cells; and the two kinds of cells are used as seed cells, the collagen sponge is taken as a bracket, and the tissue-engineered atrioventricular node with a pacing characteristic and an electrocardio-conduction characteristic is constructed in vitro. Through animal experiments, the successfully constructed tissue-engineered atrioventricular node is transplanted to treat atrioventricular conduction block, and a good treatment effect is achieved. The tissue-engineered atrioventricular node which has the electrocardio-conduction characteristic and can be transplanted for treatment is obtained, and hope is brought to treatment of arrhythmia caused by heart atrioventricular conduction block.
Description
Technical field
The present invention relates to biomedical engineering technology field, be specifically related to a kind of tissue-engineered atrioventricular node that is used for the treatment of heart atrioventricular block disease and preparation method thereof.
Background technology
The thinking of current biological pacing therapy atrioventricular block is to utilize the method for gene transfection or cell transplantation (as interventricular septum or left and right ventricles wall) below conduction block position to set up a new stable pacemaker, provide and improve idioventricular rhythm, or pace-making gene, pacemaker cell are imported to conduction block position, repair the conducting tissue of disease damage, thereby make patient avoid accepting the implantation of electronic cardiac pacemaker, reach the ideal treatment state that biological cardiac pacing is advocated.Yet although transfection pace-making genoid improves and produced the self-disciplining of myocardial cell, preliminary proof can create ectopic pacemaker, still faces huge challenge, as exogenous gene lacks in vivo expression steady in a long-term and is difficult to effective regulation and control; Can not obtain efficient, safety, carrier system that guidance quality is good etc.Utilize the method for cell transplantation to create new Pacemaker cardiac, can overcome in theory the limitation that said gene imports, the method is that the foreign cell with higher pace-making activity is transplanted to ventricle, making to form electromechanical between itself and host's myocardial cell is coupled, thereby can be used as an effective pacemaker of providing impulsion and drive ventricular systole, reach therapeutic purposes.Yet this kind of cell, owing to being transplanted to after heart the poor and source of drawing material problem of microenvironment adaptive capacity, is difficult to be applied in the future clinical treatment.(Irina Potapova, the et al.Human Mesenchymal Stem Cells as a Gene Delivery System to Create Cardiac Pacemakers.Circulation Research.2004 such as Potapova; 94:952.) mesenchymal stem cell transplantation of pace-making genetic modification is stopped in animal pattern ventricle to hole, but very easily migration, disperse in heart of the cell of transplanting can not form and stablize unified cardiac rhythm or pathway.Particularly, quiding gene and cell create new pacemaker at ventricle, try hard to improve the thinking of idioventricular rhythm treatment atrioventricular block, automatic electronic defibrillator is the same with settling, be after all a remissive treatment to symptom that conduction block produces, limited to the thorough healing effect of comprehensive improvement of cardiac function and illness.
For said gene transfection and cellular transplantation therapy the atrioventricular block problem existing and the difficulty facing, and consider the present situation of biological pacing therapy research, the atrioventricular node that external structure is engineered, it is transplanted in heart and repairs or to build cardiac conduction path again, should be the ideal solution of atrioventricular block treatment problem.
Home and abroad there is no the research report that builds tissue-engineered atrioventricular node at present, only has an example report about utilizing engineering tissue construction to set up the research of Atrioventricular Conduction path.(Choi YH, et al.2006, the Cardiac conduction through engineered tissue.Am J Pathol.2006 such as Choi; 169 (1): 72-85.) utilize Skeletal Muscle Cell and matrigel (hydrogel) to build in vitro organizer, and it is transplanted under rat heart coronary sulcus visceral pericardium, to building a house, chamber pathway has been done preliminary trial again.Result shows that this construction can set up electromechanical with host's cardiac muscle around and be coupled, and allows there is electric conduction of impulse between chamber, preliminary proof utilize the engineered feasibility of organizing the chamber pathway that builds a house again.
Summary of the invention
The object of the present invention is to provide a kind of tissue-engineered atrioventricular node and construction method thereof and application.
Inventor's imagination, utilize pacemaker cell and transfer cell as seed cell, take collagen sponge as timbering material, the atrioventricular node that external structure is engineered, it is transplanted in heart and repairs or to build cardiac conduction path again, should be the ideal solution of atrioventricular block treatment problem.The site of atrioventricular block generally occurs on the conducting path in Atrioventricular septum, and general location, about the region of Koch triangle, comprises atrioventricular node and the atrionodal region being attached thereto and end zone.In theory, tissue-engineered atrioventricular node is transplanted under the visceral pericardium at atrioventricular junction place, be about to tissue-engineered atrioventricular node and be connected in the chamber cardiac muscle of heart coronaries ditch both sides, the conduction that can cross retardance site and link up its two ends, reaches the therapeutic purposes to auriculoventricular block.
Technical scheme of the present invention is to utilize collagen sponge and mesenchymal stem cells MSCs to induce respectively Cardiac pacing cell and the transfer cell of acquisition, builds tissue-engineered atrioventricular node.
Key of the present invention is how to adopt mesenchymal stem cells MSCs to induce respectively the Cardiac pacing cell of acquisition and transfer cell as seed cell, collagen sponge is as support, build in vitro the tissue-engineered atrioventricular node with pace-making characteristic and electrocardio transport properties, for treatment heart atrioventricular block disease provides transplant.Collagen sponge is a kind of degradable biological bracket material, in vitro under condition of culture along with the change of culture environment and the passing meeting of time atrophy and degraded gradually.And the impact of the time whether seed cell growth thereon is well also planted, concentration, mode.Therefore, the size of time, concentration, mode and the timbering material of the plantation of control seed cell, shape etc., be to utilize collagen sponge and Cardiac pacing cell and transfer cell to build the key of organizational project atrioventricular node.
The invention provides a kind of construction method of tissue-engineered atrioventricular node, concrete technical scheme is as follows:
A, with sinus node cells co-culturing, inducing mesenchymal stem cells MSCs become pacemaker cell
Bone marrow extraction inoculated and cultured obtains mesenchymal stem cells MSCs, gets sino-atrial nodal tissue and carries out former culture acquisition sinus node cells.Then utilize cell culture insert (Transwell) that sinus node cells and mesenchymal stem cells MSCs are cultivated altogether, and the 5-azacytidine that adds 10mmol/L in culture fluid is in conjunction with 10
-8the endothelin-1 of M, incubation time: 6-7 days, inducing bone mesenchymal stem cell is Cardiac pacing cell.
By inverted phase contrast microscope and transmission electron microscope observing beat situation and form, Ultrastructure Features; Utilize the expression of Immuncytochemical detection induction front and back cell HCN2, HCN4, CX30.2, CX40, CX43, CX45; And the membrane current situation of full cell patch tongs technology detection cell, the methods such as operation of recording current potential are carried out Cardiac pacing cell evaluation.
B, with left auricle myocytes co-culturing, inducing mesenchymal stem cells MSCs become cardiac conduction cell
Bone marrow extraction inoculated and cultured obtains mesenchymal stem cells MSCs, and the ear's bit organization of coring is carried out former culture and obtained left auricle myocytes.Then utilize cell culture insert (Transwell) that left auricle myocytes and mesenchymal stem cells MSCs are cultivated altogether, and apply paracrine factor endothelin-1 and neuregulin-1 NRG-1 Neu Differentiation Factor NDF glial growth factors GGF (concentration range: 1 * 10
-8m-1.5 * 10
-8m), incubation time: 6-7 days, inducing bone mesenchymal stem cell becomes cardiac conduction cell.By the detection of the labels such as gap link albumen 40, HCN2 is carried out to the evaluation of cardiac conduction cell.
C, the Cardiac pacing cell and the transfer cell structure tissue-engineered atrioventricular node that utilize collagen sponge and induction to obtain
To induce successful Cardiac pacing cell and transfer cell according to a certain percentage (1: 1～1: 2) be prepared into the mixed cell suspension (concentration range 1 * 10 of mixed cell suspension
6individual/ml-1.2 * 10
6individual/ml), adopt the sedimentation method (referring to < < tissue engineering study course > >, People's Medical Officer Press, 2001 editions.Cell suspension is dripped on timbering material, is not made it overflow and be as the criterion) plant this cell mixing (planting density: 3 * 10 to collagen sponge scaffold
5individual/cm
3-6 * 10
5individual/cm
3), periodic replacement culture fluid is cultivated 15 days under condition in vitro, until cytoskeleton complex is formed with the atrioventricular node of function, external electricity irritation detects conduction velocity.
In the construction method of above-mentioned tissue-engineered atrioventricular node, sinus node cells, left auricle myocytes, mesenchymal stem cells MSCs can be selected from dog, rat, rabbit, mice etc.
In the construction method of above-mentioned tissue-engineered atrioventricular node, collagen sponge is provided by the biological company limited of Wuxi shellfish enlightening.
In the construction method of above-mentioned tissue-engineered atrioventricular node, step C is specially:
Collagen sponge is cut into 1.5 * 1.0 * 0.2cm size, uses cobalt
60irradiate above sterilization in 12 hours standby;
Get respectively Cardiac pacing cell and transfer cell that induction obtains, add 0.25% pancreatin (Gibco company) 1ml, in 37 ℃, 5%CO
2in incubator, digest 1-2 minute, under inverted phase contrast microscope, observe, cell cell space retraction is rounded, DMEM/F12 culture medium (Gibco company) 1ml adding containing 10%FBS stops digestion, with glass dropper, blows and beats into single cell suspension, and 1200 turn low-speed centrifugal 5 minutes, abandoning supernatant, add the culture fluid containing serum, with glass dropper, blow and beat into single cell suspension, adjusting cell density is 1 * 10
6individual/ml, by two kinds of cells according to a certain percentage (1: 1～1: 2) be mixed and made into mixed cell suspension, put 4 ℃ of Refrigerator stores standby;
At the bottom of 6 well culture plates, drip the cell suspension of 50 μ l, collagen sponge is placed in to cell suspension, after sponge absorbs cell suspension, to sponge surface, drip again the cell suspension of 50 μ l, be placed in 37 ℃, containing 5%CO
2incubator in hatch 1 hour, then add 2ml to contain the fresh medium of 10%FBS in culture hole, continue to be placed in 37 ℃, 5%CO
2incubator in cultivate 2 weeks, within every 2 days, change a not good liquor.
The present invention also provides the tissue-engineered atrioventricular node preparing according to said method.
The present invention also provides the application of above-mentioned tissue-engineered atrioventricular node in preparation treatment atrioventricular block disease transplant.
Through zoopery, the tissue-engineered atrioventricular node transplantation treatment of the present invention atrioventricular block successfully constructing is obtained to good result.
Pass through transplant operation, the tissue-engineered atrioventricular node successfully constructing is transplanted to heart right fibrous trigone, microsurgery is sewed up, and after two weeks and one month, causes atrioventricular block to transplanting animal, and electrocardiogram monitoring tissue-engineered atrioventricular node improves the situation of Atrioventricular Conduction.
The present invention has obtained a kind ofly to be had pace-making characteristic and electrocardio transport properties, can be used for the tissue-engineered atrioventricular node that treatment is transplanted, for cardiac arrhythmia due to the atrioventricular block for the treatment of heart has brought hope.
The specific embodiment
Now in conjunction with the embodiments, the present invention is described in detail, but enforcement of the present invention is not limited only to this.
Embodiment 1: induction Os Canitis bone marrow-drived mesenchymal stem is prepared heart pacemaker cell and transfer cell
1. separation, cultivation Os Canitis bone marrow-drived mesenchymal stem, dog sinus node cells and dog left auricle myocytes
(1) separation of Os Canitis bone marrow-drived mesenchymal stem, cultivation
The direct adherent method of the full bone marrow of dog is cultivated mesenchymal stem cells MSCs: extract hybridization dog (The 2nd Army Medical College Experimental Animal Center, 15 kilograms) the Iliac Bone 10ml that grows up, the centrifugal 10min of 900 * g, abandons supernatant and fat; Add erythrocyte cracked liquid TrisNH
4cl, 4 ℃ of 5min remove erythrocyte, with 1 * 10
6/ ml is inoculated in the coated culture dish of fibronectin (FN) (Sigma company), and culture medium is 60%DMEM-F12 (Gibco company), 10%FCS (Gibco company), 10ng/ml EGF (cytolab company), 1000U/ml LIF (Chemicon company).After 24h, remove the not attached cell that suspends, within every 3 days, change liquid 1 time, until need to go down to posterity.
(2) Neonatal Canine sinus node cells is cultivated
The dog heart of birth 24h, under anatomic microscope, in boundary's ridge middle part venous sinus side, vena cava anterior root Qu Quan sino-atrial nodal tissue; Be cut into the fragment of 1mm size, 4 ℃ of digested overnight in the pancreatin 0.07% (Gibco company).Primary Digestive system discards, then adds fresh 0.07% pancreatin, and 37 ℃ digest 15 minutes, and sucking-off Digestive system is also ended digestion, repeats 2～3 times, until all piece of tissue digests completely.After Digestive system is filtered with 200 object stainless steel filtering nets, the centrifugal 8min of 1000rpm, adds the DMEM-F12 culture medium (Gibco company) containing 20%FBS (Gibco company), 37 ℃ of 5%CO
2cultivate 4 hours, sucking-off contains the not culture fluid of attached cell cultivates in 6 orifice plates, adjusts cell number to 1 * 10
5/ ml cell, every hole inoculation 2ml.Cultivate and change liquid after 48 hours.Every hole adds 2ml to contain the DMEM-F12 culture medium of 0.1mmol/L BrdU and 20%FBS.
(3) separation and Culture of dog left auricle myocytes: get Neonatal Canine auricle muscular tissue piece; Be cut into the fragment of 1mm size, 4 ℃ of digested overnight in the pancreatin 0.07% (Gibco company).Primary Digestive system discards, then adds fresh 0.07% pancreatin, and 37 ℃ digest 15 minutes, and sucking-off Digestive system is also ended digestion, repeats 2-3 time, until all piece of tissue digests completely.After Digestive system is filtered with 200 object stainless steel filtering nets, the centrifugal 8min of 1000rpm, adds the DMEM-F12 culture medium (Gibco company) containing 20%FBS (Gibco company), 37 ℃ of 5%CO
2cultivate 4 hours, sucking-off contains the not culture fluid of attached cell cultivates in 6 orifice plates, adjusts cell number to 1 * 10
5/ ml, every hole inoculation 2ml.Cultivate and change liquid after 48 hours.The DMEM-F12 culture medium that every hole adds 2ml to contain 0.1mmol/L BrdU (Sigma company) and 20%FBS continues to cultivate.
2. mesenchymal stem cells MSCs is to the induction differentiation of Cardiac pacing cell and transfer cell direction
(1) mesenchymal stem cells MSCs is to induction differentiation and the evaluation of Cardiac pacing cell direction
Mesenchymal stem cells MSCs adds Transwell cell, and density is every hole 1 * 10
4individual cell.The Transwell cell of repopulating cell is inserted in 6 orifice plates of growth sinus node cells, at 37 ℃ of 5%CO
2in incubator, cultivate.After 24 hours, apply the 5-azacytidine (Sigma) of 10mmol/L in conjunction with 10
-8the endothelin-1 of M (Tocris), continuous culture is carried out the detection of inducing cell and is identified after 7 days.
Inverted phase contrast microscope can be observed beating of individual cells, and cellular morphology major part is fusiformis and triangle.Immuncytochemical detection found that the expression of cell HCN2, HCN4, CX30.2, CX45 (Abcam company) is positive; Full cell patch tongs technology detects the action potential of individual cells, shows and has spontaneous depolarization diastasis, proves that the rear cell of induction is pacemaker cell.
(2) mesenchymal stem cells MSCs is to induction differentiation and the evaluation of heart transfer cell direction
Mesenchymal stem cells MSCs adds Transwell cell, and density is every hole 1 * 10
4individual cell.The Transwell cell of repopulating cell is inserted in 6 orifice plates of growth left auricle myocytes, at 37 ℃ of 5%CO
2in incubator, cultivate.After 24 hours, to above-mentioned six orifice plates and Transwell cell, add paracrine factor endothelin-1 (Tocris) and neuregulin-1 NRG-1 Neu Differentiation Factor NDF glial growth factors GGF (RD company), making its final concentration is 10
-8m, continuous culture is carried out the detection of inducing cell and is identified after 7 days.
Inverted phase contrast microscope can be observed beating of individual cells, and cellular morphology major part is fusiformis and triangle.The expression of cell inserted by connexin 40 (CX40) and HCN2 (Abcam company) before and after Immuncytochemical detection induction, result shows that the positive expression of CX40 and HCN2 obviously raises (p < 0.05), shows that mesenchymal stem cells MSCs transforms to heart transfer cell direction.Full cell patch tongs technology detects the action potential of individual cells, shows and has spontaneous depolarization diastasis, proves that the rear cell of induction is transfer cell.
Embodiment 2: the dog Cardiac pacing cell that induction obtains and transfer cell are planted collagen sponge and built tissue-engineered atrioventricular node
1. the structure of tissue-engineered atrioventricular node
(1) collagen sponge is prepared
Collagen protein derives from the type i collagen of cattle heel string, and collagen sponge is provided by the biological company limited of Wuxi shellfish enlightening.Collagen sponge is cut into 1.5 * 1.0 * 0.2cm size, uses cobalt
60irradiate above sterilization in 12 hours standby.
(2) dog Cardiac pacing cell and transfer cell mixed liquor inoculation collagen sponge
Get respectively Cardiac pacing cell and transfer cell that induction obtains, add 0.25% pancreatin (Gibco company) 1ml, in 37 ℃, 5%CO
2in incubator, digest 1-2 minute, under inverted phase contrast microscope, observe, cell cell space retraction is rounded, DMEM/F12 culture medium (Gibco company) 1ml adding containing 10%FBS stops digestion, blows and beats into single cell suspension, centrifugal 5 minutes of 1000rpm with glass dropper, abandoning supernatant, add the culture fluid containing serum, with glass dropper, blow and beat into single cell suspension, adjusting cell density is 1 * 10
6individual/ml, by two kinds of cells according to a certain percentage (1: 1～1: 2) be mixed and made into mixed cell suspension, put 4 ℃ of Refrigerator stores standby.At the bottom of 6 well culture plates, drip the mixed cell suspension of 50 μ l, collagen sponge is placed in to cell suspension, after sponge absorbs cell suspension, to sponge surface, drip again the mixed cell suspension of 50 μ l, be placed in 37 ℃, containing 5%CO
2incubator in hatch 1 hour, then add the fresh culture fluid that contains 10% hyclone of 2ml in culture hole, continue to be placed in 37 ℃, containing 5%CO
2incubator in cultivate 2 weeks, within every 2 days, change a not good liquor.
2. the detection of tissue-engineered atrioventricular node is identified
(1) Cardiac pacing cell and the growth of transfer cell on collagen sponge are observed in HE dyeing
The above-mentioned growth Cardiac pacing cell of 2 weeks, transfer cell and collagen sponge complex are prepared into paraffin section and carry out HE dyeing, step is as follows: haematoxylin liquid dyes 10min, tap water rinses, 75% ethanol solution hydrochloride differentiation 30s, washing 1h, 95% ethanol 1～2min, 1～2min is redyed in Yihong, 95% ethanol 1～2min, dewaters transparent, neutral gum mounting.By HE dye in observation of cell collagen sponge complex seed cell growth evenly, contact closely, Cardiac pacing cell and transfer cell and collagen sponge are compound well.
(2) optics potential detector (Optical mapping) and lead pace-making characteristic and the conduction velocity that physiograph detects cell collagen sponge complex more
Use optics potential detector the pacemaker potential of collagen sponge complex can be detected, lead physiograph more and detect cell collagen sponge complex conduction velocity, result shows that the conduction velocity of electricity impulsion is 0.014 ± 0.001m/s, similar to body atrioventricular nodal region current potential and conduction velocity, prove that constructed cell collagen sponge complex (tissue-engineered atrioventricular node) has the characteristic of similar heart atrioventricular node.
Embodiment 3: in tissue-engineered atrioventricular node transplant, observe conducting effect
By open chest surgery, the above-mentioned tissue engineering conduction bundle successfully constructing is transplanted to dog (The 2nd Army Medical College Experimental Animal Center, 15 kilograms) under the visceral pericardium at atrioventricular junction place, be about to tissue engineering conduction bundle and be connected in the chamber cardiac muscle of heart coronaries ditch both sides, microsurgery is sewed up; Transplant and within first 3 days, start to apply ciclosporin A (Beijing Shuanglu Pharmaceutical Co., Ltd., dosage is 0.25mg/ days) and prednisolone (Shanghai General Pharmaceutical Co., ltd., dosage is 0.1mg/ days) Immunosuppression rejection.
Result shows that organizational project atrioventricular node of the present invention can survive in host's cardiac muscle, and the cell in transplanted tissue and host's myocardial cell have formed electromechanical Rhizoma Nelumbinis connection; Transplant and to animal, make atrioventricular block after one month, electrocardiogram (Alcott biological company limited) monitoring display organization through engineering approaches atrioventricular node can improve between chamber and conducts, and confirms that constructed tissue-engineered atrioventricular node has the potential of building again heart Atrioventricular Conduction path.
Claims (4)
1. a construction method for tissue-engineered atrioventricular node, comprises the steps:
A, with sinus node cells co-culturing, inducing mesenchymal stem cells MSCs become pacemaker cell
Bone marrow extraction inoculated and cultured obtains mesenchymal stem cells MSCs, get sino-atrial nodal tissue and carry out former culture acquisition sinus node cells, then utilize cell culture insert that sinus node cells and mesenchymal stem cells MSCs are cultivated altogether, and the 5-azacytidine that adds 10mmol/L in culture fluid is in conjunction with 10
-8the endothelin-1 of M, incubation time 6-7 days, inducing bone mesenchymal stem cell is Cardiac pacing cell;
B, with left auricle myocytes co-culturing, inducing mesenchymal stem cells MSCs become cardiac conduction cell
Bone marrow extraction inoculated and cultured obtains mesenchymal stem cells MSCs, the ear's bit organization of coring is carried out former culture and is obtained left auricle myocytes, then utilize cell culture insert that left auricle myocytes and mesenchymal stem cells MSCs are cultivated altogether, and apply paracrine factor endothelin-1 and neuregulin-1 NRG-1 Neu Differentiation Factor NDF glial growth factors GGF, concentration is 1 * 10
-8m-1.5 * 10
-8m, incubation time 6-7 days, inducing bone mesenchymal stem cell becomes cardiac conduction cell;
C, the Cardiac pacing cell and the transfer cell structure tissue-engineered atrioventricular node that utilize collagen sponge and induction to obtain
Collagen sponge is cut into 1.5 * 1.0 * 0.2cm size, uses cobalt
60irradiate above sterilization in 12 hours standby;
Get respectively Cardiac pacing cell and transfer cell that induction obtains, add 0.25% pancreatin 1ml, in 37 ℃, 5%CO
2in incubator, digest 1-2 minute, under inverted phase contrast microscope, observe, cell cell space retraction is rounded, the DMEM/F12 culture medium 1ml adding containing 10%FBS stops digestion, with glass dropper, blows and beats into single cell suspension, and 1200 turn low-speed centrifugal 5 minutes, abandoning supernatant, add the culture fluid containing serum, with glass dropper, blow and beat into single cell suspension, adjusting cell density is 1 * 10
6individual/ml, is mixed and made into mixed cell suspension by two kinds of cells by 1:1～1:2, puts 4 ℃ of Refrigerator stores standby;
At the bottom of 6 well culture plates, drip the cell suspension of 50 μ l, collagen sponge is placed in to cell suspension, after sponge absorbs cell suspension, to sponge surface, drip again the cell suspension of 50 μ l, be placed in 37 ℃, containing 5%CO
2incubator in hatch 1 hour, then add 2ml to contain the fresh medium of 10%FBS in culture hole, continue to be placed in 37 ℃, 5%CO
2incubator in cultivate two weeks, within every two days, change a not good liquor.
2. the construction method of tissue-engineered atrioventricular node according to claim 1, is characterized in that wherein said sinus node cells, left auricle myocytes, mesenchymal stem cells MSCs derive from dog, rat, rabbit or mice.
3. the tissue-engineered atrioventricular node preparing according to the arbitrary construction method of claim 1 to 2.
4. a tissue-engineered atrioventricular node as claimed in claim 3 is treated the application in atrioventricular block disease transplant in preparation.
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| CN103705984B (en) * | 2013-12-17 | 2016-06-15 | 南京大学医学院附属鼓楼医院 | Collagen scaffold combined with mesenchymal stem cells preparation method and application |
| CN104232571B (en) * | 2014-01-29 | 2018-01-12 | 中国人民解放军第二军医大学 | A kind of construction method of engineered beating tissue |
| CN105911096B (en) * | 2016-03-29 | 2018-07-10 | 南京艾尔普再生医学科技有限公司 | A kind of artificial heart system that can carry out drug pharmacological toxicology screening in vitro |
| DE102016223423B4 (en) * | 2016-11-25 | 2018-06-07 | Universität Rostock | Method, device and kit for the determination of cardiac conduction |
| TWI643952B (en) * | 2017-10-31 | 2018-12-11 | 胡瑜峰 | Methods and compositions for generating pacemaker cells |
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| US20040214182A1 (en) * | 2003-04-25 | 2004-10-28 | Vinod Sharma | Genetic modification of targeted regions of the cardiac conduction system |
| US20070005134A1 (en) * | 2005-06-27 | 2007-01-04 | The Cleveland Clinic Foundation | Apparatus for placement in the annulus of a tricuspid valve |
| CN101100655A (en) * | 2007-06-15 | 2008-01-09 | 中国人民解放军第二军医大学 | Method for orienting inducing and differentiating heart pacemaker cell by embryo source pluripotent stem cell |
| CN101415458A (en) * | 2003-11-10 | 2009-04-22 | 辛弗尼医药有限公司 | Method to control ventricular rate in atrial fibrillation patients |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040214182A1 (en) * | 2003-04-25 | 2004-10-28 | Vinod Sharma | Genetic modification of targeted regions of the cardiac conduction system |
| CN101415458A (en) * | 2003-11-10 | 2009-04-22 | 辛弗尼医药有限公司 | Method to control ventricular rate in atrial fibrillation patients |
| US20070005134A1 (en) * | 2005-06-27 | 2007-01-04 | The Cleveland Clinic Foundation | Apparatus for placement in the annulus of a tricuspid valve |
| CN101100655A (en) * | 2007-06-15 | 2008-01-09 | 中国人民解放军第二军医大学 | Method for orienting inducing and differentiating heart pacemaker cell by embryo source pluripotent stem cell |
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