| Objective:1) To obtain recombinant adenovirus (rAd) mediated sarcoplasmic reticulum Ca2+ATPase (SERCA2a) gene with high titer for meeting the needs of the concentration and quantity of gene transfection. To establish stably and efficiently methods for amplification and purification of SERCA2a gene and the basis of gene therapy research of heart failure (HF).2) This study will use bone marrow stem cell (BMSC) transplantation therapy, SERCA2a gene therapy and SERCA2a gene-modified BMSC transplantation therapy methods on the basis of acute myocardial infarction (AMI) animal models by the histopathological technology, non-invasive cardiac electrical technology, ultrasound technology, tissue electrophysiological techniques compare the differences and advantages of the three methods of treatment effect, discuss the therapy effectiveness of BMSC in AMI and SERCA2a in HF after MI, and evaluate the feasibility of BMSC as SERCA2a gene carrier strategy for short-term effective treatment. This study focus on improving cardiac function, preventing myocardial infarction after heart failure and ventricle remodeling. The key point is to study electro-mechanical coupling and electrophysiological characteristics in SERCA2a therapy/BMSC therapy/SERCA2a gene-modified BMSC transplantation therapy and to leading/reducing/preventing arrhythmia and to provide clinical basis for cell transplantation-based gene therapy in AMI. Methods:1) Human embryonic kidney cells (HEK-293) were transfected by adenovirus mediated SERCA2a gene with100μl at virus concentration of1.9x1012pfu/ml, when the cells appeared cytopathic effect (CPE), collected and destroyed cell wall with freeze-thaw steps to deliver virus, got the Ad-SERCA2a-GFP products by2×CsCl-gradient purification and measured DNA plasmids by the ultraviolet spectrophotometer.2) Twenty-six adult male SD rats were randomly divided into three groups:sham group (n=10), rAd.β-gal group (n=8) and rAd.SERCA2a group (n=8). Sham operation consisted of thoracotomy and cardiac exposure without coronary artery ligation. rAd.β-gal group and rAd.SERCA2a group were ligated the left anterior descending coronary artery for HF rats after MI, while transfecting β-gal and SERCA2a gene into the hearts respectively. We used ultrasound electrocardiogram for evaluating cardiac diastolic and systolic function, electrocardiogram (ECG) monitoring and microelectrode arrays (MEA) technology for myocardium electrical activity in vitro.3) Rats with myocardial infarction (n=24) and divided into3groups randomly:saline group (sham group, n=8), BMSC transplantation group (BMSC group, n=8) and SERCA2a gene modified BMSC transplantation group (BMSC+rAd.SERCA2a group, n=8). After2week, cardiac function was evaluated by echocardiography and heart electrical activity was evaluated by ECG and MEA technology.4) We used38rats to set up AMI model (n=38) and divided into5groups randomly:SERCA2a gene group (rAd.SERCA2a group, n=8), BMSC transplantation group (BMSC group, n=8), SERCA2a gene modified BMSC transplantation group (BMSC+rAd.SERCA2a group, n=8), saline group (sham group, n=8) and rAd.lacZ group (n=7). After2week, cardiac function was evaluated by echocardiography, morphological changes of infarction tissue was by assessed HE staining and heart electrical activity was evaluated by ECG and MEA technology. Results:1) Green fluorescence were successfully expressed in HEK-293. The infectious titer, the number of virus particle and the ratio of infectious titer to virus particle for the product were1.3±0.58×1012pfu/ml, The OD260/OD280ratio was1.57±0.49(n=50).2) SERCA2a and β-gal gene carried by rAd were successfully transfected in heart failure rats. In rAd.SERCA2a group failing heart function were improved. Compared with the sham group, In rAd.SERCA2a group the ventricular end diastolic volume and left ventricular end-systolic volume[(0.410±0.130) cm2.vs.(0.39±0.02) cm2,(0.08±0.02) cm2.vs.(0.06±0.01) cm2, P>0.05] were increased, left ventricular ejection fraction[(82.3±4.59)%.vs.(85.56±1.26)%, P>0.05] and fractional shortening[(46.6±2.32)%.vs.(49.58±1.71)%, P>0.05] were no changes. Compared with the sham group, QT interval was prolonged [(111.02±7.42) ms.vs.(94.7±1.55) ms, n=6, P<0.05)] in ECG, and the incidence rate of premature ventricular contractions (PVC) was71.5%(5/7) in rAd.β-gal group, but in rAd.SERCA2a group QT interval shortened [(81.45±4.97) ms.vs.(94.7±1.55) ms, n=6, P<0.05]and the incidence rate of PVC was14.3%(1/7). No significant difference in the heart rate of rAd.SERCA2a group[(435±31) bpm.vs.(442 ±22) bpm, n=6, P>0.05] by MEA records. However, compared with the rAd.p-gal group, the maximum field potential, the minimum field potential and field potential duration were prolonged [(0.82±0.39) mV. vs.(0.64±0.13) mV,(1.88±0.57) mV. vs.(1.35±0.12) mV,(124.17±21.08) ms. vs.(113.23±12.02) ms, respectively, n=6, P<0.05] in rAd.SERCA2a group. The field potential duration were statistically different between the infarct zone and the contralateral normal zone [(60.36±2.08) ms.vs.(103.24±7.35) ms, n=5, P<0.05] in rAd.β-gal group, and field potential duration dispersion in infarct zone with60channels record was larger than rAd.SERCA2a group[(38.5±4.62) ms.vs.(26.88±5.09) ms, n=5]. The conduction time was simultaneous in rAd.SERCA2a group, and the cardiac electroconduction activity could keep consistency and improve in myocardial infarction tissue.3) The transduction ration of rAd.SERCa2a to BMSCs were80%to90%.Compared to sham group, BMSC group and BMSC+rAd.SERCA2a group could significantly increase left ventricular ejection fraction on14days after therapy (n=6, P<0.05). QT duration of BMSCs+rAd.SERCA2a group was significantly shorter than in the sham group[(80.30±6.53) ms.vs.(105.31±21.89) ms], the mean value was significantly decreased23.8%(±=7, P<0.05). Premature ventricular contractions (PVCs) were also recorded in rats from sham group. MEA results suggested that the maximum field potential and field potential duration of infarcted myocardium area in BMSC group and BMSC+rAd.SERCA2a group were significantly longer than those in sham group[the maximum field potential:(0.51±0.15) mV,(0.55±0.16) mV,(0.23±0.1) mV; field potential duration:(104.5±25.43) ms,(107.67±24.01) ms,(63±20.34) ms, n=6,P<0.05]. The conduction time was the shortest in BMSC+rAd.SERCA2a group, the cardiac electrical conduction could keep consistency and improve in myocardial infarction tissue.4) Compared with shame group[(70.49±3.27)%], rAd.SERCA2a group[(82.63±2.58)%], BMSC group[(80.24±4.15)%] and BMSC+rAd.SERCA2a group[(84.28±2.46)%] significantly improved cardiac ejection fraction (P<0.05, n=6), but rAd.LacZ group [(71.34±2.42)%] was not statistically significant (P>0.05,n=5). ECG can be found that QT interval in rAd.SERCA2a group [(83.07±17.56) ms, n=6] and BMSCs+rAd.SERCA2a group [(81.20±5.64) ms, n=7) significantly decreased by18.9%and20.7%(P<0.05) by compared with shame group [(102.42±20.67) ms, n=7]. Shame group and rAd.lacZ group also appeared more PVCs. MEA results suggested that isolated heart beats were significantly slowed and ventricular arrhythmias and atrioventrocular block were recorded in sham group. The field potential duration (FPdur) of isolated heart tissue in rAd. SERCA2a group (121.25±18.64) ms, BMSC group (106.12±20.76) ms and BMSC+rAd. SERCA2a group (106.35±19.51) ms (n=6) was significantly longer than shame group [(60.45±19.12) ms, n=6](P<0.05), rAd. SERCA2a group significantly increased field potentials and improved electrical activities conduction in infracted tissue. The degeneration and necrosis of infracted myocardium can be were prevented in rAd. SERCA2a group and BMSC+rAd. SERCA2a group. Conclusion:The conclusion of the study can be drawn from the following four parts,1) High titer of rAd.SERCA2a were provided for gene therapy of cardiovascular research. The reliably experimental method was established on amplification of rAd.SERCA2a by HEK-293, it has certainly referred values for the amplification and purification of recombination adenovirus carrying other genes.2) BMSC is an effective transporter of gene therapy.3) Overexpression of adenovirus mediated SERCA2a gene in the short-term could effectively enhance heart function, increase heart beats and significantly improve electrical activities conduction and reduce arrhythmia in infarction heart.4) SERCA2a, BMSC and SERCA2a gene modified BMSC transplantation could obviously enhance cardiac function.But overexpression of SERCA2a could reduce necrosis of myocardium and delay ventricular remodeling. rAd.SERCA2a group could the most effectively increase field potential and improve electrical conduction of infarcted myocardium, block the occurrence of arrhythmia after myocardial infarction.5) MEA technology is an ideal technology for observing rhythm, frequency and conduction activities in cardiovascular disease animal models. MEA technology have important value of cells or gene therapy in the cardiac electrical activity area. |
PDF Full Text Request