Method for regulating and controlling conduction velocity of tissue-engineered conduction bundle and application
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A tissue engineering and conduction velocity technology, applied in the field of biomedical engineering, can solve problems such as slowing myocardial conduction velocity
Pending Publication Date: 2022-08-05
THE NAVAL MEDICAL UNIV OF PLA
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Bakker et al. further overexpressed Tbx3 in cardiomyocytes in vivo and in vitro, and found that Tbx3 reset the gene program of mature cardiomyocytes, inhibited the expression of fast conduction channels such as Connexin43, Connexin40, Nav1. Martijn LBakker, Gerard J J Boink, Bas J Boukens, et al. T-box transcription factor TBX3 reprogrammes mature cardiac myocytes into pacemaker-like cells. Cardiovasc Res. 2012 Jun 1; 94(3):439-49.)
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Embodiment 1
[0044] Example 1. Screening of the regulation scheme of rat ECT conduction velocity
[0045] 1. TBX3 transfected H9C2 cells
[0046] (1) Design PCR primers, extract total RNA from rat embryos, amplify Tbx3 cDNA, insert Tbx3 cDNA into pLenti-EF1-EGFP-P2A-Puro-CMV-3Flag-WPRE to obtain pLenti-EF1-EGFP-P2A- The Puro-CMV-TBX3-3Flag-WPRE recombinant vector system was co-cultured with pHelper1.0 and pHelper2.0;
[0047] (2) Discard the original medium in the culture bottle, add 3ml PBS to wash three times, add 3ml trypsin and place in a 37°C constant temperature incubator to digest for 3min;
[0048] (3) Observe under the microscope, when the digestion is complete, mix with a pipette, add 20ul to the cell counting plate, and count with a cell counter;
[0049] (4) Centrifuge with a centrifuge, discard the supernatant, add 1 ml of complete medium and mix well, and pipette the cell suspension into a 6-well plate, so that the number of cells in each well is 10 6 indivual;
[0050] (...
Embodiment 2
[0078] Example 2: Verification of the effect of ECT in vivo transplantation after conduction velocity regulation
[0079] The ECT successfully constructed in the above example 1 was transplanted into the subepicardium at the atrioventricular junction of rats (Experimental Animal Center, Second Military Medical University, 250 g) through thoracotomy, that is, the ECT was connected to the atrioventricular sulcus on both sides of the heart. In myocardium; cyclosporine A (Beijing Shuanglu Pharmaceutical Co., Ltd., dose of 0.25 mg / day) and prednisolone (Shanghai General Pharmaceutical Co., Ltd., dose of 0.1 mg / day were started 3 days before transplantation ) to suppress immune rejection.
[0080] Animals were anesthetized for 6 weeks after transplantation, atrioventricular block was performed, and electrocardiogram monitoring was performed to verify conduction velocity that produced physiological atrioventricular delay. ECT was adjusted using the obtained parameters, and the adjus...
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Abstract
The invention provides a method for regulating and controlling the conduction velocity of a tissue-engineered conduction bundle and application. By changing the cell planting density and the tissue culture period in the tissue engineering heart conduction bundle construction and culture process, it is found that the conduction speed is regulated and controlled most effectively when the planting density is 1 * 10 < 7 >-1.1 * 10 < 7 > cells / ml and the culture period is 2 weeks. On the basis of the parameters, heart cells overexpressing Tbx3 and common heart progenitor cells are jointly planted on a collagen sponge stent to construct ECT, and it is found that the effect of precise regulation and control of the conduction velocity is better through the matching of 70-80% of Tbx3 + H9C2 cells and 20-30% of Tbx3-H9C2 cells. The conduction velocity of the ECT constructed by the method is close to the in-vivo atrioventricular junction level, the key problem of realizing physiological atrioventricular electrical conduction in the process of reconstructing an atrioventricular pathway through a tissue engineering conduction bundle (ECT) is solved, namely, the problem of how to ensure atrioventricular extension after ECT transplantation is solved, and an effective method is provided for regulating and controlling the conduction velocity of the ECT.
Description
technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to a method for regulating the conduction velocity of a tissue-engineered conduction bundle for treating heart atrioventricular block disease and a tissue-engineered conduction bundle constructed by the method. Background technique [0002] Severe atrioventricular block is a common complication of various diseases in cardiac surgery, which seriously endangers human health. At present, it is mainly treated by implanted electronic pacemaker. Electronic pacemakers can improve the symptoms of patients, but due to their mechanical structure, there are many problems such as susceptibility to electromagnetic interference, lack of neurohumoral regulation, electrode infection, battery replacement, and inability to develop with random body development. In view of the insufficiency of electronic pacemakers, engineered conduction tract or tissue (ECT), as a new idea for the treat...
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