CN114854693A

CN114854693A - Method for regulating and controlling conduction speed of tissue engineering conduction bundle and application

Info

Publication number: CN114854693A
Application number: CN202210419511.1A
Authority: CN
Inventors: 张喜
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2022-04-21
Filing date: 2022-04-21
Publication date: 2022-08-05

Abstract

The invention provides a method for regulating and controlling the conduction speed of a tissue engineering conduction bundle and application thereof. The invention discovers that the planting density is 1 multiplied by 10 by changing the cell planting density and the tissue culture period length in the tissue engineering cardiac conduction bundle construction culture process ⁷ cells/ml～1.1×10 ⁷ cell/ml, 2 weeks of culture cycle, was most effective in regulating conduction velocity. In this context the parametersBased on the fact that ECT is constructed by planting heart cells over expressing Tbx3 and common heart progenitor cells in combination in a collagen sponge scaffold, 70-80% of Tbx3 is found ⁺ H9C2 cells with 20-30% Tbx3 ^‑ The H9C2 cell ratio has better effect on accurately regulating and controlling the transmission speed. The ECT conduction velocity constructed by the invention is close to the junction level of the body chamber, the problem that the realization of physiological chamber electric conduction is very critical in the process of reconstructing a chamber access by a tissue engineering conduction bundle (ECT), namely the problem of how to ensure the chamber delay after the ECT is transplanted is solved, and an effective method is provided for the regulation and control of the ECT conduction velocity.

Description

Method for regulating and controlling conduction speed of tissue engineering conduction bundle and application

Technical Field

The invention relates to the technical field of biomedical engineering, in particular to a method for regulating and controlling the conduction speed of a tissue engineering conduction bundle for treating heart atrioventricular block diseases and the tissue engineering conduction bundle constructed by the method.

Background

Severe atrioventricular block is a common complication of various diseases in cardiology and surgery, seriously harms human health, and is mainly treated clinically at present by a method of implanting an electronic pacemaker. The electronic pacemaker can improve the symptoms of patients, but has the problems of susceptibility to electromagnetic interference, lack of neurohumoral regulation, electrode infection, battery replacement, incapability of developing along with the development of organisms and the like due to a mechanical structure. In view of the deficiency of the electronic pacemaker, the tissue Engineered cardiac conduction tract (ECT) has gradually gained acceptance and attention of scholars at home and abroad as a new idea for treating atrioventricular block. ECT is a method of tissue engineering to construct a bundle of "conducting tissues" between the atrioventricular chambers, and the "conducting tissues" are used to communicate and block the conduction between the atrioventricular chambers. The idea is tissue transplantation regeneration treatment, can overcome the defects of an electronic pacemaker theoretically, and is an ideal solution for the atrioventricular block treatment problem.

Currently, studies have preliminarily confirmed that ECT can reestablish atrioventricular pathways, but to achieve physiologic atrioventricular electrical conduction, a very critical problem must be solved, namely how to ensure atrioventricular delay after ECT transplantation. Normal atrioventricular conduction is delayed by the atrioventricular node in the atrioventricular junction area, and the atrioventricular myocardium can orderly contract to ensure the completion of the blood pumping function. For example, the conduction velocity of human atrioventricular node is very low, only 0.02m/s, so the excitation is slowly conducted at the atrioventricular junction to generate delay, and the delay time is about 0.1 s. However, the conduction speed of the ECT reported at present is far higher than the level at the atrioventricular junction of the body (0.02-0.05m/s), so that the ECT cannot delay signals after being transplanted like the normal atrioventricular junction, and the atrioventricular cavity is difficult to excite and coordinate to contract in sequence. In order to solve the problem, the regulation and control of the ECT conduction speed must be considered, however, the research at home and abroad is lack of the deep discussion of the work.

The main structural factor affecting the high and low conduction speed in the heart is the expression distribution of gap junction protein. For example, human sinoatrial and atrioventricular nodes are mainly composed of P-cells and T-cells, and the expression is mainly Connexin45 and Connexin30.2, which have low conductivity, so that the conduction velocity (sinoatrial node is 0.05m/s, and atrioventricular node is about 0.02m/s) is slower than that of other tissues of the cardiac conduction system. The atrioventricular bundle and the left and right bundle branches mainly comprise T cells and Purchase cells, the number of the Purchase cells expressing more gap junction proteins is obviously more, and the types of the gap junction proteins mainly comprise Connexin40 and Connexin43 with high conductivity, so that the conduction speed of the atrioventricular bundle and the left and right bundle branches is higher and reaches 1.2-2 m/s. In addition, the atrial muscle is mainly Connexin40, and the ventricular muscle is mainly Connexin43, so the working myocardium is also fast-conducting. Therefore, the amount and type of gap junction protein expression in different parts of the heart are directly related to the conduction speed.

The transcription factor Tbx3 is one of the members of T-box family, widely exists in embryonic tissues and is involved in the development and control of a plurality of important organs in the embryonic period. It is closely related to cardiac conduction system development, inhibits fast-conducting Connexin40 and Connexin43, initiates activation of slow-conducting Connexin45 and Connexin30.2 in cardiac progenitor cells, and regulates the final differentiation maturation of the nodal cell phenotype. Based on the above-mentioned developmental regulation of Tbx3, researchers attempted to directly manipulate the gene over-expression and directed induction of cardiomyocyte differentiation towards desmocyte phenotype. Hoogaars et al injected the Tbx3 gene into the right atrium of rats and found that ectopic expression of Tbx3 inhibited Connexin40, activated Connexin30.2, and transformed atrial muscle to a desmoid type (Willem M.H. Hoogaars, Angela Engel, Janynke F.Brons, et al. Tbx3 controls the lateral node gene program and improses compressor function on the atria. genes Dev.2007May 1; 21(9): 1098-. Bakker et al further overexpressed Tbx3 in cardiomyocytes in vivo and in vitro, and found that Tbx3 reprogrammed the gene of mature cardiomyocytes, inhibited the expression of fast conduction channels such as Connexin43, Connexin40, Nav1.5, and resulted in a decrease in myocardial conduction velocity (Martijn LBaker, Gerard J Boink, Bas J Boukens, et al.T-box transcription factor TBX3 reprogrammes materials cardiac myocytes inter-pacemaker-like cells, Cardiovasc Res.2012Jun 1; 94(3): 439-49.).

In conclusion, Tbx3 has the regulatory effects of inhibiting fast-conducting and activating slow-conducting phenotype in cardiac progenitors and mature myocardium. The transcription factor is an important potential regulation point of the gap junction expression of the ECT and can be applied to the regulation of the ECT conduction speed. Overexpression of Tbx3 in cardiac progenitors, inhibition of expression of fast-conducting channels in ECT, activation of initiation of slow-conducting gap junctions, makes it possible to set ECT conduction velocity to a lower level, enabling physiologic atrioventricular conduction for ECT transplantation therapy. Therefore, the Tbx3 is taken as a control point, and the conduction speed of the ECT can be effectively controlled by combining the cell planting density and the culture period.

Disclosure of Invention

The invention is based on the research, and aims to provide a method for regulating and controlling the conduction velocity of a tissue engineering conduction bundle so as to construct ECT with the conduction velocity close to the junction level of a human body chamber. The invention also aims to provide application of the ECT obtained by regulating and controlling the conduction velocity of the tissue engineering conduction bundle, and the constructed ECT is transplanted to the epicardium of the atrioventricular groove of a complete atrioventricular conduction block animal to reconstruct an atrioventricular conduction pathway, so that the effect of ECT transplantation on treating atrioventricular conduction block diseases is verified and debugged.

The first purpose of the invention is to provide a method for regulating and controlling the conduction velocity of a tissue engineering conduction bundle, wherein the planting density of 1 multiplied by 10 is found by regulating and controlling the cell planting density and the length of a tissue culture period in the process of constructing and culturing the tissue engineering cardiac conduction bundle ⁷ cells/ml～1.1×10 ⁷ cell/ml, 1 week of culture cycle, was most effective in regulating conduction velocity. On the basis of the parameter, heart cells over expressing the Tbx3 and common heart progenitor cells are planted in combination to construct the ECT on the collagen sponge scaffold, and the expression quantity of the Tbx3 on the construct is controlled according to different preparation ratios of the two types of cells; after detection, 70-80% of Tbx3 is found ⁺ H9C2 cells with 20-30% Tbx3 ^- The H9C2 cell ratio has good effect on accurately regulating and controlling the transmission speed.

By separately detecting Tbx3 ⁺ And Tbx3 ^- The expression of Bmp2, Alk3 and Smad in the ECT shows that Tbx3 and a Bmp2 signal channel have mutual regulation and control relationship,by activating this signaling pathway, there is a role in the regulation of ECT conduction velocity.

According to the invention, the ECT with the conduction speed close to the interface level of the body chamber is constructed by utilizing the construction parameters, the Tbx3 and the relation between the signal molecules and the conduction speed; and transplanting the constructed ECT to the lower part of the atrioventricular groove epicardium of the complete atrioventricular conduction block animal to establish an atrioventricular conduction pathway, and verifying and debugging the effect of the ECT transplantation on treating the atrioventricular conduction block disease.

The invention provides a method for regulating and controlling the conduction velocity of a tissue engineering conduction bundle, which is specifically designed as follows:

1. establishing an H9C2 cell model for over-expressing Tbx3

Designing a PCR primer, extracting rat embryo total RNA, amplifying Tbx3 cDNA, inserting the Tbx3 cDNA into pLenti-EF1-EGFP-P2A-Puro-CMV-3Flag-WPRE to obtain a pLenti-EF1-EGFP-P2A-Puro-CMV-TBX3-3Flag-WPRE recombinant vector system; further co-transfecting 293T cells with pHelper1.0 and pHelper2.0 to determine the lentivirus titer; and (3) transfecting cells by using the complete lentivirus stable transfection system to establish an H9C2 cell model for over-expressing Tbx 3.

For PCR primer sequences see table 1 below:

TABLE 1 Tbx3 amplification primer sequences

2. Planting Tbx3 overexpression model cells on collagen sponge scaffold to construct ECT

Cutting the collagen sponge scaffold into small pieces with size of 10mm 3mm, and using pre-Co ⁶⁰ Irradiating for more than 12 hr, and sterilizing.

At 70-80% Tbx3 ⁺ H9C2 cells with 20-30% Tbx3 ^- H9C2 cell proportioning, namely planting Tbx3 overexpression model cells on a collagen sponge scaffold to construct ECT, wherein the planting density ranges from 1 × 107cells/ml to 1.1 × 107cells/ml, the in-vitro culture period of the scaffold is 1 week, and the specific process is as follows:

(1) before planting, the treatment process of Tbx3 overexpression recombinant cells is as follows: adding 0.25% pancreatin to37℃、5％CO ₂ Digesting for 1-2 minutes in an incubator, observing under an inverted phase contrast microscope, enabling cell bodies to retract to be circular, adding DMEM/F12 culture medium containing 10% FBS to stop digestion, blowing and beating into single cell suspension by using a glass dropper, centrifuging at low speed for 5 minutes at 1200 rpm, removing supernate, adding culture solution containing serum, blowing and beating into single cell suspension by using a glass dropper, adjusting the cell density to be 1-1.1 multiplied by 10 ⁷ Storing in a refrigerator at 4 deg.C for use;

(2) the method for planting the Tbx3 overexpression recombinant cells comprises the following steps: tbx3 overexpressing Tbx3 ⁺ H9C2 recombinant cells and Tbx3 ^- The H9C2 recombinant cells are proportioned in proportion to prepare cell suspension with corresponding planting density; slowly dripping 150 mu L of the cell suspension onto the bracket by using a 200 mu L pipette, and culturing for 4 hours in a constant-temperature incubator at 37 ℃ after the cell suspension slowly permeates;

(3) the in vitro culture method of ECT is as follows: dropping a certain amount of cell suspension into the porous culture plate, placing the ECT into the cell suspension, absorbing the cell suspension by the bracket sponge, dropping the same amount of cell suspension onto the sponge surface, placing at 37 deg.C, and adding 5% CO ₂ The culture box is incubated for 1 hour, then a fresh culture solution containing 10% fetal calf serum is added into the culture hole, and the culture hole is continuously placed at 37 ℃ and contains 5% CO ₂ The culture chamber was maintained for 1 week, and the medium was changed every 2 days.

The culture solution containing 10% fetal calf serum comprises the following components: 10% FBS, 89% DMEM/F12 medium, 1% penicillin streptavidin (Gibco Co.).

The second purpose of the invention is to provide the tissue engineering conduction bundle constructed according to the regulation and control method and carry out corresponding detection.

(1) The conduction velocity of the constructed ECT is detected by using an MPA multi-channel electrophysiology instrument and a speed measuring device, the integrity and continuity of tissue cells of the constructed object are detected by HE staining, and the expressions of gap junction proteins Connexin40, Connexin45, Connexin43, pacing genes HCN2, HCN4 and alphaActin are detected by immunofluorescence chemical staining and PCR.

And (3) detecting conduction velocity results by using a multi-lead physiological instrument to display: based on 70-80% Tbx3 ⁺ H9C2 and20-30％Tbx3 ^- the ECT conduction speed of the H9C2 mixture is obviously lower than that of the mixture based on Tbx3 ^- ECT (p) of H9C2<0.05), proving that the conduction speed of the constructed ECT is effectively regulated and controlled and the method is feasible; HE staining shows that the ECT H9C2 cells grow uniformly and are in close contact with each other, and extracellular matrix of the cell and the scaffold complex is remodeled, which indicates that the ECT has a structural basis for exerting functional activity; immunofluorescence chemical staining and PCR detection show that: HCN2, HCN4, Connexin45 and AlphaActin all expressed positively, but based on 70-80% Tbx3 ⁺ H9C2 with 20-30% Tbx3 ^- Connexin45 and AlphaActin expression intensity in ECT prepared from H9C2 is more obvious (p)<0.05), indicating that a slow conduction phenotype has been established that modulates conduction velocity.

(2) Verifying the function of the Bmp2 signal in the regulation of ECT conduction velocity by Tbx3

PCR (polymerase chain reaction) is carried out to detect the expression of Bmp2, Alk3, Smad and Tbx3 in the constructed ECT, the pathway inhibitor LDN-193189 and the pathway activator Kartogenin are respectively added to verify the mutual regulation and control relation of the signal pathways of Tbx3 and Bmp 2; bmp2 was added to H9C2 cells and collagen sponge scaffold complex (cultured for 1 week), conduction velocity of ECT constructed was measured by MPA multichannel electrophysiology apparatus and speed measuring device, integrity and continuity of tissue cells of construct were measured by HE staining, gap junction protein Connexin40, Connexin45, Connexin43, pacing gene HCN2, HCN4, alphaActinin expression were measured by immunofluorescence chemical staining and PCR.

PCR detection shows that: after the Bmp2 is added, Alk3 and Smad are increased, and the conduction speed is reduced; HE and immunofluorescence chemical staining also show that Bmp2 has the function based on Tbx3, after Bmp2 is added, extracellular matrix of cells and a scaffold complex is well remodeled, the expression of HCN2, HCN4, Connexin45 and AlphaActin is positive, the expression intensity of Connexin45 and AlphaActin is more obvious (p <0.05), and the function can be regulated by a pathway inhibitor LDN-193189 and a pathway activator Kartogenin. The result shows that the Bmp2 signal can regulate Tbx3 and is an effective target point for ECT conduction velocity regulation.

(3) Performance debugging and effect verification of ECT (acute lymphoblastic leukemia) transplantation atrioventricular block animal model

Constructing ECT with conduction velocity close to the junction level of human body atrioventricular by the method, transplanting the constructed ECT to the epicardial membrane of the interatrioventricular groove of the animal, constructing an atrioventricular conduction path, and monitoring electrocardiogram every 1 week; after transplantation, animals are respectively anesthetized at each time phase point (2 weeks, 4 weeks and 8 weeks), atrioventricular conduction block is made, atrioventricular electric conduction is monitored through electrocardiogram, and the conduction speed of physiological atrioventricular delay is verified; taking out the tissue of the transplanted area at each time phase point, and detecting the integrity and continuity of the cell construction in the transplanted tissue by the HE; and (3) debugging the ECT by using the obtained parameters, using the debugged ECT for transplantation treatment of atrioventricular block, and discussing the treatment effect of the ECT on the atrioventricular block diseases through histological and electrophysiological detection.

The results show that: after transplantation, the atrioventricular conduction is blocked, the survival rate is obviously improved by transplanting the ECT within one week after the block, histological detection shows that the ECT and the myocardium of the host have the expression of gap connexin, and the continuity and the integrity are better, and electrocardiogram shows that no priming syndrome occurs in animals. The results show that the transplanted tissue can survive in the myocardium of the host, and the cells in the transplanted tissue and the cells in the myocardium of the host form electromechanical coupling; after the conduction speed is regulated and controlled, the ECT in vivo transplantation effect is good.

The invention has the following beneficial guarantee and effects:

the invention discovers that the planting density is 1 multiplied by 10 by changing the cell planting density and the tissue culture period length in the tissue engineering cardiac conduction bundle construction culture process ⁷ cells/ml～1.1×10 ⁷ cell/ml, 2 weeks of culture cycle, was most effective in regulating conduction velocity. On the basis of the parameter, heart cells over expressing Tbx3 are planted in a combined mode (Tbx 3) ⁺ H9C2) and common cardiac progenitors (Tbx 3) ^- H9C2) in collagen sponge scaffold to construct ECT, and 70-80% of Tbx3 is found ⁺ H9C2 cells with 20-30% Tbx3 ^- The H9C2 cell ratio has better effect on accurately regulating and controlling the transmission speed.

The ECT conduction velocity constructed by the invention is close to the junction level of the atrioventricular system of a body, and animal model experiments show that after the ECT is transplanted into a rat, transplanted tissues can survive in host cardiac muscle, cells in the transplanted tissues and the host cardiac muscle cells form electromechanical coupling, and monitoring shows that the ECT regulated and controlled by the conduction velocity can realize physiological atrioventricular delay, thereby providing a new choice for the treatment of severe atrioventricular conduction block.

Therefore, the invention solves the critical problem of realizing physiological atrioventricular electric conduction in the process of reconstructing an atrioventricular pathway by using a tissue engineering conduction bundle (ECT), namely the problem of how to ensure the atrioventricular delay after the ECT is transplanted, and provides an effective method for regulating and controlling the ECT conduction speed.

Drawings

FIG. 1 is a comparison of 2 ratios of ECT morphologies observed under a fluoroscope: a is based on 80% Tbx3 ⁺ H9C2+20％Tbx3 ^- ECT of H9C 2; b is based on 0% Tbx3 ⁺ H9C2+100％Tbx3 ^- ECT of H9C 2.

FIG. 2 is a graph of HE staining based on 80% Tbx3 ⁺ H9C2+20％Tbx3 ^- ECT cells of H9C2 complexed with the scaffold.

FIG. 3 shows the expression of functional molecules of heart observed by immunofluorescence chemical staining: a is based on 80% Tbx3 ⁺ H9C2+20％Tbx3 ^- Connexin45 for H9C2 ECT; b is based on 0% Tbx3 ⁺ H9C2+100％Tbx3 ^- Connexin45 for H9C2 ECT; c is based on 80% Tbx3 ⁺ H9C2+20％Tbx3 ^- Alpha actin of H9C2 ECT; d is 0% Tbx3 ⁺ H9C2+100％Tbx3 ^- AlphaActinin from H9C2 ECT.

FIG. 4 is HE staining to observe the complexing of ECT cells with scaffolds based on BMP induction.

FIG. 5ECT in vivo transplantation assay: a is the site of ECT transplantation at the atrioventricular junction of the heart (ra is the right atrium, rv is the right ventricle, graft site), B is the electrocardiographic test, based on 0% Tbx3 ⁺ H9C2+100％Tbx3 ^- ECT transplantation with H9C2 presents with a pre-excitation syndrome (indicated by the arrow), C is an electrocardiogram test based on 80% Tbx3 ⁺ H9C2+20％Tbx3 ^- ECT transplantation of H9C2 did not present pre-stress syndrome.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The experimental procedures, in which specific conditions are not specified, in the following examples are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Percentages and parts are by volume unless otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

EXAMPLE I screening of the ECT transduction Rate control protocol in rats

TBX3 transfected H9C2 cells

(1) Designing a PCR primer, extracting rat embryo total RNA, amplifying Tbx3 cDNA, inserting the Tbx3 cDNA into pLenti-EF1-EGFP-P2A-Puro-CMV-3Flag-WPRE to obtain pLenti-EF1-EGFP-P2A-Puro-CMV-TBX3-3Flag-WPRE recombinant vector system, and co-culturing the recombinant vector system with pHelper1.0 and pHelper2.0;

(2) removing the original culture medium in the culture bottle, adding 3ml PBS for washing three times, adding 3ml trypsin, and placing in a constant temperature incubator at 37 ℃ for digestion for 3 min;

(3) observing under a microscope, mixing uniformly by using a suction pipe after complete digestion, sucking 20ul of the mixture, adding the mixture into a cell counting plate, and counting by using a cell counter;

(4) centrifuging with centrifuge, removing supernatant, adding 1ml complete culture medium, mixing, sucking cell suspension into 6-well plate to make cell number of 10 per well ⁶ A plurality of;

(5) culturing in a 37 deg.C constant temperature incubator for 24 hr, removing culture medium, washing with PBS for three times, adding 1ml trypsin for digestion for 3min, sucking 20 μ l, adding into a cell counting plate, and counting with a cell counter to obtain cell number per well;

(6) determining MOI to be 80 through multiple transfection preliminary experiments, calculating the virus amount required by each group, and preparing 750ul virus suspension by using a DMEM (DMEM) culture medium (Gibco company) preheated by 37 ℃ in a water bath kettle; adding the virus suspension into a 6-hole plate, placing the 6-hole plate into a constant-temperature incubator at 37 ℃, shaking the plate once every half hour, adding 750 mu l of complete culture medium into each hole after 2 hours, and placing the plate into the constant-temperature incubator at 37 ℃ for continuous culture;

(7) after 12 hours, the complete medium was replaced with fresh medium, and transfection efficiency was observed by a fluorescence microscope at 72 hours, and puromycin screening was performed.

2. TBX 3-transfected H9C2 cell-seeded sponge scaffold

(1) Mixing Tbx3 ⁺ H9C2 cells and Tbx3 ^- H9C2 cells were treated with 80% Tbx3 cells, respectively ⁺ H9C2+20％Tbx3 ^- H9C2 cell fraction, 70% Tbx3 ⁺ H9C2+30％Tbx3 ^- H9C2 cell fraction, 0% Tbx3 ⁺ H9C2+100％Tbx3 ^- Different proportions of H9C2 cells were tested to prepare 1X 10 cells ⁷ ～1.1×10 ⁷ cell/ml cell suspension for later use;

(2) sterilizing collagen sponge scaffold with size of 10mm x 3mm by ray, placing into 6-well plate, slowly dripping 150 μ l of the above cell suspension onto the sponge scaffold by using 200 μ l of pipette gun, and culturing in 37 deg.C constant temperature incubator for 4 hr after the cell suspension slowly permeates;

(3) adding 2ml of complete culture medium into each hole, culturing in a constant temperature incubator at 37 ℃, observing and recording the shape of the sponge bracket every day, changing the culture solution 1 time every 2 days, and planting and culturing for 1 week.

Comparative results referring to FIG. 1, fluorescence microscopy based on 80% Tbx3 ⁺ H9C2+20％Tbx3 ^- The ECT cells of H9C2 have uniform growth, tight connection, good activity and strong positive fluorescence; based on 70% Tbx3 ⁺ H9C2+30％Tbx3 ^- The observed results for ECT cells of H9C2 were also similar; based on 0% Tbx3 ⁺ H9C2+100％Tbx3 ^- The ECT of H9C2 only obscured visible fluorescence (fig. 1).

3. Confirmation and detection of ECT characteristics of cytoskeletal complex

The cell proportion is 80 percent of Tbx3 according to the better experimental result ⁺ H9C2+20％Tbx3 ^- H9C2 or 70% Tbx3 ⁺ H9C2+30％Tbx3 ^- The ECT characteristics were tested under the H9C2 test conditions.

3.1 Using MPA multichannel electrophysiology apparatus and speed measuring device to detect the conduction velocity of the cytoskeleton complex

Taking the ECT to be tested out of the culture medium, washing away the water on the surface by using filter paper to prevent lead, placing the ECT vertical to the electrode wire, and carrying out the electric conduction test with the stimulation amplitude of 0.5ms and the hardware filtering range of 40-1200 Hz. The electric conduction speed is obtained by calculating the ratio of the distance (delta d) between the two channel electrodes to the time difference delta t of the wave crest of the electric conduction wave.

The result of detecting the conduction velocity by using the multi-lead physiological instrument shows that: based on 70-80% Tbx3 ⁺ H9C2 with 20-30% Tbx3 ^- The ECT conduction speed of the H9C2 mixture is obviously lower than that of the mixture based on Tbx3 ^- ECT (p) of H9C2<0.05) and proves that the conduction velocity of the constructed ECT is effectively regulated and the method is feasible.

3.2 histological examination

(1) Adding 10ml PBS into the cell scaffold complex, placing the cell scaffold complex in a shaking table for 1 hour, and repeating twice; removing clear water, adding 10ml of 4% paraformaldehyde, fixing for 12 hours in a shaking table, and dehydrating a 30% sucrose solution to a bottom; freezing for 6 hr, slicing with a freezing microtome to a thickness of 5um, sticking on glass slide, oven drying at 37 deg.C, and storing in a refrigerator at-20 deg.C;

(2) HE staining examined the integrity and continuity of the cytoskeletal complex: taking out the slices from a refrigerator at 4 ℃, melting wax, and dewaxing to water conventionally; staining with hematoxylin for 10 min, washing with distilled water, differentiating with 75% ethanol hydrochloride differentiation solution for 3 s, and washing with tap water to turn blue; re-dyeing with eosin for 40 seconds, washing with water to remove redundant colors, performing microscopic examination, and decoloring with 75% alcohol if the color is too heavy until the color is proper; drying at room temperature, transparent xylene, and sealing with neutral gum. HE staining showed 80% Tbx3 ⁺ H9C2+20％Tbx3 ^- The ECT cells of H9C2 grew uniformly and were in intimate contact, and the extracellular matrix of the cell and scaffold complex was remodeled, suggesting that ECT has a structural basis for functional activity (FIG. 2).

(3) And (3) carrying out immunofluorescence chemical staining detection:

a) washing with PBS for 5min × 3, sealing with normal sheep serum sealing solution (1:10), and standing at 37 deg.C for 1 hr;

b) adding primary antibodies (gap junction protein Connexin40, Connexin45, Connexin43, pacemaker gene HCN2, HCN4 and alphaActinin), and incubating at 4 deg.C overnight;

c) washing with PBS for 5min × 3, adding goat anti-rabbit IgG-FITC (1:100), incubating at 37 deg.C for 1 h;

d) PBS wash 5min × 3, DAPI (1 μ g/m1, diluted with 0.01mol/L PBS), room temperature 3 min;

e) washing with water for 5min × 3, sealing with fluorescence attenuation resistant sealing agent, and observing under a fluorescence microscope.

Immunofluorescence chemical staining and PCR detection show that: HCN2, HCN4, Connexin45 and AlphaActin are positive in expression, but Connexin45 and AlphaActin are more significant in expression intensity (p <0.05) in ECT based on 70-80% of Tbx3+ H9C2 and 20-30% of Tbx3-H9C2, and a slow conduction phenotype for regulating and controlling conduction speed is established (FIG. 3).

3.3 Effect of Bmp2 Signaling in the modulation of the transduction velocity of the above-described cytoskeletal Complex by Tbx3

(1) PCR and Westernblot were used to detect the expression of Bmp2, Alk3 and Smad in the above cell-scaffold complex;

(2) respectively adding the pathway inhibitor LDN-193189 and the pathway activator Kartogenin, and verifying the mutual regulation and control relationship between the Tbx3 and the Bmp2 signal pathway;

(3) bmp2 was added to H9C2 cells and collagen sponge scaffold complex for 2 weeks, MPA multichannel electrophysiology apparatus and speed measuring device were used to detect conduction velocity of ECT constructed, HE staining was used to detect integrity and continuity of tissue cells of constructs, transmission electron microscopy was used to detect sub-micro structure of ECT tissue cells, immunofluorescence chemical staining was used to detect gap junction proteins Connexin45, Connexin30.2, Connexin40, Connexin43, ion channel Na (V)1.5, etc.

PCR detection shows that: after the Bmp2 is added, Alk3 and Smad are increased, and the conduction speed is reduced; HE and immunofluorescence chemical staining also show that Bmp2 has the function based on Tbx3, the extracellular matrix of cells and a scaffold complex is well remodeled after Bmp2 is added (figure 4), the expressions of HCN2, HCN4, Connexin45 and AlphaActin are all positive, the expression intensities of Connexin45 and AlphaActin are more obvious (p is less than 0.05), and the function can be regulated by a pathway inhibitor LDN-193189 and a pathway activator Kartogenin. The result shows that the Bmp2 signal can regulate Tbx3 and is an effective target point for ECT conduction velocity regulation.

Example two: ECT in vivo transplantation effect verification after conduction velocity regulation

The ECT successfully constructed in the first example above was implanted under the ventricle of the atrioventricular junction of rats (center of laboratory animals, 250 g, second university of military medical science) by an open chest surgery, i.e., the ECT was ligated into the atrioventricular myocardium on both sides of the coronary sulcus of the heart; cyclosporin a (beijing biont pharmaceutical industry gmbh, dosage 0.25 mg/day) and prednisolone (shanghai tong pharmaceutical industry gmbh, dosage 0.1 mg/day) were applied to suppress immune rejection 3 days before transplantation.

After transplantation, the animals were anesthetized at 6 weeks, atrioventricular conduction blocks were made, and electrocardiogram monitoring was performed to verify the physiological atrioventricular conduction velocity. And (3) debugging the ECT by using the obtained parameters, using the debugged ECT for transplantation treatment of atrioventricular block, and discussing the treatment effect of the ECT on the atrioventricular block diseases through histological detection.

After transplantation, the atrioventricular conduction is blocked, the survival rate is obviously improved by transplanting the ECT within one week after the block, histological detection shows that the ECT and the myocardium of a host have the expression of gap junction protein, the continuity and the integrity are better, and electrocardiogram shows that no priming syndrome occurs in animals (figure 5).

The present invention is not limited to the embodiments described above, and those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are included in the scope defined by the claims of the present application.

Sequence listing

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ctttgtagtc ggatccaggg gacccgctgc aggacc 36

Claims

1. A method for regulating and controlling the conduction velocity of a tissue engineering conduction bundle is characterized by comprising the following steps:

tbx3 overexpressing Tbx3 ⁺ H9C2 recombinant cells and Tbx3 ^- The H9C2 recombinant cells are mixed according to a specific proportion and planted on a bracket to construct ECT, and the planting density range is 1 multiplied by 10 ⁷ cells/ml～1.1×10 ⁷ cells/ml, culture cycle 1 week, construction to obtain conduction velocity near the atrioventricular junction level of ECT.

2. The method for regulating and controlling the conduction velocity of the tissue engineering conduction bundle according to claim 1, wherein:

wherein, Tbx3 ⁺ H9C2 recombinant cells and Tbx3 ^- The H9C2 recombinant cells are planted according to the proportion of 70-80% to 20-30%.

3. The method for regulating and controlling the conduction velocity of the tissue engineering conduction bundle according to claim 1, wherein:

wherein the scaffold is collagen sponge scaffold with size of 10mm 3mm, and pre-used Co ⁶⁰ Irradiating for more than 12 hr, and sterilizing.

4. The method for regulating and controlling the conduction velocity of the tissue engineering conduction bundle according to claim 1, wherein:

the construction method of the Tbx3 overexpressed H9C2 recombinant cell comprises the following steps:

after the Tbx3 cDNA is amplified, the Tbx3 cDNA is inserted into pLenti-EF1-EGFP-P2A-Puro-CMV-3Flag-WPRE to obtain a pLenti-EF1-EGFP-P2A-Puro-CMV-TBX3-3Flag-WPRE recombinant vector system; further co-transfecting the recombinant vector system with pHelper1.0 and pHelper2.0 to 293T cells, and determining the titer of lentivirus; cells were transfected with the completed lentivirus stable transfection system to create H9C2 recombinant cells overexpressing Tbx 3.

5. The method for regulating and controlling the conduction velocity of the tissue engineering conduction bundle according to claim 1, wherein:

before planting, the treatment process of the Tbx3 overexpression recombinant cells is as follows: adding 0.25% pancreatin at 37 deg.C and 5% CO ₂ Digesting for 1-2 minutes in an incubator, observing under an inverted phase contrast microscope, enabling cell bodies to retract to be circular, adding DMEM/F12 culture medium containing 10% FBS to stop digestion, blowing and beating into single cell suspension by using a glass dropper, centrifuging at low speed for 5 minutes at 1200 rpm, removing supernate, adding culture solution containing serum, blowing and beating into single cell suspension by using a glass dropper, adjusting the cell density to be 1-1.1 multiplied by 10 ⁷ And (4) storing each sample per ml in a refrigerator at 4 ℃ for later use.

6. The method for regulating and controlling the conduction velocity of the tissue engineering conduction bundle according to claim 1, wherein:

the method for planting the Tbx3 overexpression recombinant cells comprises the following steps: tbx3 overexpressing Tbx3 ⁺ H9C2 recombinant cells and Tbx3 ^- The H9C2 recombinant cells are proportioned in proportion to prepare cell suspension with corresponding planting density; mu.L of the cell suspension was slowly dropped onto the scaffold using a 200. mu.L pipette, and after the cell suspension was slowly infiltrated, it was cultured in a 37 ℃ incubator for 4 hours.

7. The method for regulating and controlling the conduction velocity of the tissue engineering conduction bundle according to claim 1, wherein:

the in vitro culture method of ECT comprises the following steps: dropping a certain amount of cell suspension into the porous culture plate, placing the ECT into the cell suspension, absorbing the cell suspension by the bracket sponge, dropping the same amount of cell suspension onto the sponge surface, placing at 37 deg.C, and adding 5% CO ₂ The culture box is incubated for 1 hour, then a fresh culture solution containing 10% fetal calf serum is added into the culture hole, and the culture hole is continuously placed at 37 ℃ and contains 5% CO ₂ The culture is carried out in an incubator for 2 weeks, the culture solution is changed every 2 days,

the culture solution containing 10% fetal calf serum comprises the following components: 10% FBS, 89% DMEM/F12 medium, 1% streptavidin.

8. The tissue engineering conduction bundle constructed by the regulation and control method according to any one of claims 1 to 7.