肌浆网钙ATP酶基因转导治疗急性心肌梗塞的实验研究

发布时间：2018-05-05 23:45

本文选题：肌浆网钙ATP酶 + 心肌梗塞　；参考：《新疆医科大学》2013年博士论文

【摘要】：目的：1)通过扩增和纯化重组腺病毒携带肌浆网钙ATP酶(rAd.SERCA2a)基因,获得高效价的rAd.SERCA2a基因,可满足基因转染浓度和数量的需要,同时也为扩增和纯化肌质网Ca2+-ATP酶(SERCA2a)基因提供一种稳定高效的实验方法,也为心力衰竭(HF)的基因治疗研究提供基础。2)本研究是建立在急性心肌梗塞(AMI)动物模型基础上,分别采用经典的骨髓干细胞(BMSC)治疗、SERCA2a基因治疗以及SERCA2a基因修饰的BMSC移植(BMSC+SERCA2a)治疗方法,运用组织病理技术,无创心电技术,超声技术,组织细胞电生理技术,对比三种方法治疗效果的差异性和优越性,分别探讨BMSC治疗AMI、转SERCA2a基因治疗心肌梗塞后心力衰竭的有效性,以及两者联合治疗的可行性,评价BMSC作为基因载体策略的可行性。本研究在改善心脏功能,纠正心梗后心力衰竭,防止心室重塑方面进行研究,重点探讨国、内外研究都比较薄弱的环节即：转SERCA2a基因治疗/BMSC移植治疗/BMSC+SERCA2a治疗AMI后的心脏电一机械匹配和电生理特性的改变,导致/减少/防止心律失常的发生进行评估,为以细胞移植为基础的基因治疗AMI提供临床应用基础。方法：1)复苏和传代人胚肾细胞(HEK-293),用100μl1.9×1012pfu/ml rAd.SERCA2a感染HEK-293细胞,出现细胞病变效应时收获细胞,经物理反复冻融方法及两步氯化铯超速离心方法获得纯化的rAd.SERCA2a,紫外分光光度计比色法测定病毒DNA质粒数。2)将急性心肌梗塞后心力衰竭动物模型制作成功的26只成年雄性SD大鼠随机分为3组：假手术组(n=10),空病毒对照组(rAd.β-gal组,n=8)和SERCA2a基因转染组(rAd.SERCA2a组,n=8)。假手术组仅开胸不结扎冠状动脉,rAd.β-gal组和rAd.SERCA2a组分别进行左冠状动脉前降支结扎建立大鼠心肌梗塞后心力衰竭动物模型,同时分别将携带β-gal和SERCA2a基因的重组腺病毒导入衰竭心脏,术后2周超声心电图检测心脏的舒张和收缩功能,心电图监测体表心电活动,微电极阵列(MEA)监测离体心脏组织电活动情况。3)建立成年雄性SD大鼠AMI模型(n=24)。随机分为3组：盐水对照组(对照组,n=8),BMSC移植组(BMSC组,n=8),SERCA2a基因修饰的BMSC移植组(BMSC+rAd.SERCA2a组,n=8)。术后2周进行心脏B超、体表心电图及MEA技术评估心功能和心脏电活动变化。4)制作成年雄性SD大鼠AMI动物模型(n=38)。随机分为5组：SERCA2a基因组(rAd.SERCA2a组,n=8),BMSC移植组(BMSC组,n=8),SERCA2a基因修饰的BMSC移植组(BMSC+rAd.SERCA2a组,n=8),盐水对照组(shame组,n=7),腺病毒空载体组(rAd.LacZ组,n=7)。术后2周记录体表心电图以及运用心脏二维超声评价心功能,HE染色评估心脏组织形态改变,运用MEA技术评估离体心脏组织电活动情况。结果：1)SERCA2a基因在HEK-293细胞成功呈绿色荧光表达,纯化的rAd.SERCA2a.GFP DNA质粒数为1.3±0.58×1012pfu/ml, OD260/OD280比值为1.57±0.49(n=50)。2)rAd携带SERCA2a与β-gal基因均成功转入大鼠衰竭心脏。Ad.SERCA2a组可改善心功能,与假手术组相比心室舒张末期容积与心室收缩末期容积有轻微的增加[(0.410±0.130) cm2对(0.39±0.02) cm2,(0.08±0.02) cm2对(0.06±0.01) cm2, P0.05]、左心室射血分数[(82.3±4.59)%对(85.56±1.26)%,P0.05]和短轴缩短率[(46.6±2.32)%对(49.58±1.71)%,P0.05]无明显改变。与假手术组相比,rAd.β-gal组体表心电图QT间期延长[(111.02±7.42)ms对(94.7±1.55)ms,n=6,P0.05],室性早搏发生率达71.5%(5/7),而rAd.SERCA2a组QT间期缩短[(81.45±4.97)ms对(94.7±1.55) ms,n=6, P0.05],室性早搏发生率达14.3%(1/7)。MEA记录可发现rAd.SERCA2a组心率与假手术组相比差异无统计学意义[(435±31)bpm对(442±22) bpm, n=6, P0.05],与rAd. β-gal组相比,rAd.SERCA2a组最大场电位[(0.82Q0.39)mV对(0.64±0.13)mV,n=6,P0.05]、最小场电位[(1.88±0.57)mV对(1.35±0.12) mV, n=6,P0.05]、场电位时限[(124.17±21.08)ms对(113.23±12.02) ms, n=6, P0.05]均延长；rAd.β-gal组梗死区与梗死对侧区心肌组织场电位时限差异有统计学意义[(60.36±2.08)ms对(103.24±7.35) ms, n=5, P0.05],并且60通道记录梗死区心肌组织场电位时程离散度大于rAd.SERCA2a组[(38.5ms±4.62) ms对(26.88±5.09) ms, n=5]; rAd.SERCA2a组传导基本一致,使心肌梗塞面心室肌组织电活动呈均一性传导。3)rAd.SERCA2a基因对BMSC的感染效率为80%～90%。BMSC组、BMSC+rAd.SERCA2a组大鼠左室射血分数明显优于对照组(n=6,P0.05)。BMSC+rAd.SERCA2a组大鼠体表心电图QT间期为(80.30±6.53)ms比对照组(105.31±21.89)ms少了23.8%(P0.05),对照组室性早搏较多。MEA记录可发现梗死面心肌组织最大场电位BMSC组为(0.51±0.15)mV、BMSC+rAd.SERCA2a组为(0.55±0.16)mV,均明显高于对照组的(0.23±0.1)mV(P均0.05),场电位时程BMSC组为(104.5±25.43)ms、BMSC+rAd.SERCA2a组为(107.67±24.01)ms,均显著长于对照组的(63±20.34)ms(P均0.05)。BMSC+rAd.SERCA2a组传导时间最短,且能显著改善心梗面心室肌组织的均一性传导。4)rAd.SERCA2a组[(82.62±2.58)%]、BMSC组[(80.24±4.15)%]、BMSC+rAd.SERCA2a组[(84.28±2.46)%]与shame组[(70.49±3.27)%]相比射血分数均有明显改善(P0.05,n=6),而rAd.LacZ组[(71.34±2.42)%]与shame组相比差异没有统计学意义(P0.05,n=5)。体表心电图可发现与shame组[(102.42±20.67)ms,n=7]相比,QT间期在rAd.SERCA2a组[(83.07±17.56)ms,n=6]和BMSC+rAd.SERCA2a组[(81.20±5.64)ms,n=7]均值明显减少18.9%和20.7%(P0.05),在shame组和rAd.LacZ组也出现较多的室性早搏。MEA记录可发现对照组离体心脏搏动频率明显减慢,并出现室性心律失常和房室传导阻滞。离体心脏组织场电位时程(FPdur)在rAd.SERCA2a组(121.25±18.64) ms BMSC组(106.12±20.76)ms和BMSC+rAd.SERCA2a组(106.35±19.51)ms(n=6)显著长于shame组[(60.45±19.12) ms, n=6](P0.05),其中rAd.SERCA2a组较显著地增加心梗区心室肌场电位及减少心律失常发生。rAd.SERCA2a组和BMSC+rAd.SERCA2a组能够从组织形态上阻止梗死心肌发生变性和坏死。结论：1)本研究建立了稳定可靠的借助HEK-293细胞培养扩增rAd.SERCA2a的实验方法,纯化后的高效价的SERCA2a基因的重组腺病毒可直接用于心血管疾病的动物实验研究,对重组腺病毒携带其他基因的扩增与提纯方法也具有一定的参考价值。2)BMSC是一种有效的基因治疗运载体。3)腺病毒载体介导的SERCA2a基因过度表达能够在短期内有效地增强心脏功能,增加梗死心脏的搏动频律和显著改善梗死心肌的电传导和减少心律失常发生。4)SERCA2a基因、BMSC移植以及SERCA2a基因修饰BMSC移植均可以明显改善心肌梗塞后心脏功能,但过表达SERCA2a基因可以减少心肌坏死和延缓心室重构,rAd.SERCA2a组可以更有效地增加心梗区心室肌场电位、改善心梗面心室的均一性传导,并能够预防心梗后心律失常的发生。5)MEA技术是一项检测心血管疾病动物模型心脏组织电生理节律和频率以及传导活动的理想技术。MEA技术在细胞或基因治疗的心脏电活动研究方面具有重要的应用价值。
[Abstract]:Objective: 1) by amplification and purification of the recombinant adenovirus carrying sarcoplasmic reticulum calcium ATP (rAd.SERCA2a) gene, a highly efficient rAd.SERCA2a gene can be obtained to meet the needs of the gene transfection concentration and quantity. At the same time, a stable and efficient method for amplification and purification of the sarcoplasmic reticulum Ca2+-ATP enzyme (SERCA2a) gene is also provided, and also for heart failure (HF). Based on the animal model of acute myocardial infarction (AMI), this study is based on the animal model of acute myocardial infarction (AMI), using classical bone marrow stem cells (BMSC), SERCA2a gene therapy and SERCA2a gene modified BMSC transplantation (BMSC+SERCA2a), using histopathology, noninvasive electrocardiography, ultrasound technology, and tissue. Cell electrophysiological techniques, compared with the differences and advantages of the three methods of treatment, respectively explore the effectiveness of BMSC treatment of AMI, SERCA2a gene therapy for heart failure after myocardial infarction, and the feasibility of the combination of the two treatments, and evaluate the feasibility of the BMSC as a gene carrier strategy. Stress failure to prevent ventricular remodeling. The focus of the study is to explore the weak links in both domestic and foreign studies. The transfer of SERCA2a gene therapy to /BMSC transplantation for the treatment of cardiac electrical mechanical matching and electrophysiological changes after /BMSC+SERCA2a treatment for AMI, and the assessment of the occurrence of / decrease / prevent arrhythmia, for cell transplantation Basic gene therapy AMI provides a basis for clinical application. Methods: 1) resuscitation and generation of human embryonic kidney cells (HEK-293), infected HEK-293 cells with 100 micron l1.9 x 1012pfu/ml rAd.SERCA2a, and harvested cells in the presence of cytopathic effects. The purified rAd.SERCA2a is obtained by physical repeated freezing and thawing methods and two steps of cesium chloride overspeed centrifugation. 26 adult male SD rats of acute myocardial infarction after acute myocardial infarction were randomly divided into 3 groups: sham operation group (n=10), empty virus control group (rAd. beta -gal group, n=8) and SERCA2a gene transfection group (rAd.SERCA2a group, n=8). The sham operation group had not ligation of the coronary artery in the sham operation group, and the rA was not ligation of the coronary artery. The D. beta -gal group and the rAd.SERCA2a group were ligation of the left anterior descending branch of the coronary artery to establish the rat model of heart failure after myocardial infarction. At the same time, the recombinant adenovirus carrying the beta -gal and the SERCA2a gene was introduced into the exhaustion heart respectively. The diastolic and contractile function of the heart was detected by the ultrasonic electrocardiogram 2 weeks after the operation, and the electrocardiogram monitoring of the body surface electrocardiogram (ECG) was monitored. Microelectrode array (MEA) was used to monitor the electrical activity of isolated cardiac tissue (.3) to establish the adult male SD rat AMI model (n=24). It was randomly divided into 3 groups: saline control group (control group, n=8), BMSC transplantation group (BMSC group, n=8), SERCA2a gene modified BMSC transplantation group (BMSC+ group,). After 2 weeks of operation, the heart B ultrasound, body surface electrocardiogram and technical evaluation were evaluated. The AMI animal model (n=38) of adult male SD rats (.4) was randomly divided into 5 groups: SERCA2a genome (rAd.SERCA2a group, n=8), BMSC transplantation group (BMSC group, n=8), SERCA2a gene modified BMSC transplantation group, saline control group, adenovirus free load group. 2 weeks after the operation, the body surface electrocardiogram was recorded and the cardiac function was evaluated by two dimensional echocardiography. HE staining was used to evaluate the changes of cardiac tissue morphology. MEA technique was used to evaluate the electrical activity of the isolated heart tissue. Results: 1) the SERCA2a gene was expressed in green fluorescence in HEK-293 cells, and the number of purified rAd.SERCA2a.GFP DNA plasmids was 1.3 + 0.58 x 1012pfu/ Ml, OD260/OD280 ratio was 1.57 + 0.49 (n=50).2) rAd carrying SERCA2a and beta -gal gene were all successfully transferred to the rat heart failure cardiac.Ad.SERCA2a group to change the heart function. Compared with the sham group, the ventricular end diastolic volume and ventricular end systolic volume were slightly increased [(0.410 + 0.130) cm2 pairs (0.39 + 0.02) cm2, (0.08 + 0.02) cm2 pairs (0.06 + 0.01). Cm2, P0.05], left ventricular ejection fraction (82.3 + 4.59)% (85.56 + 1.26)%, P0.05] and short axis shortening rate [(46.6 + 2.32)% of (49.58 + 1.71)%, P0.05] no significant change. Compared with sham operation group, rAd. beta -gal group electrocardiogram QT interval prolonged [(111.02 + 7.42) ms pairs (94.7 + 1.55) ms, n=6, P0.05], ventricular premature beat rate reached 71.5% (5/7) The QT interval of group RCA2a was shortened [(81.45 + 4.97) ms pairs (94.7 + 1.55) ms, n=6, P0.05], and the incidence of ventricular premature beat was 14.3% (1/7).MEA record. The heart rate of rAd.SERCA2a group was not statistically significant compared with that of sham operation group [435 + 31) BPM (442 + 22) BPM, n=6, and n=6. For (0.64 + 0.13) mV, n=6, P0.05], the minimum field potential [(1.88 + 0.57) mV pairs (1.35 + 0.12) mV, n=6, P0.05], and field potential time limit [(124.17 + 21.08) ms (113.23 + 12.02) ms, n=6, P0.05] were extended, and there was a statistically significant difference between the infarct area of the rAd. beta group and the myocardial group in the lateral region of the infarct [60.36 2.08) =5, P0.05], and 60 channels recorded myocardial temporal dispersion of infarct area greater than that of group rAd.SERCA2a [(38.5ms + 4.62) ms pairs (26.88 + 5.09) ms, n=5]; rAd.SERCA2a group conduction is basically consistent, the myocardial infarction surface ventricular muscle tissue electrical activity is homogeneous conduction.3) rAd.SERCA2a gene to BMSC infection efficiency is 80% ~ 90%.BMSC group The left ventricular ejection fraction of the SC+rAd.SERCA2a group was significantly better than that of the control group (n=6, P0.05). The QT interval of the body surface electrocardiogram in the group.BMSC+rAd.SERCA2a rats was (80.30 + 6.53) MS than that of the control group (105.31 + 21.89) ms less 23.8% (P0.05), and the most significant.MEA recording of the ventricular premature beat in the control group was (0.51 + 0.15) mV, BMSC (0.51 + 0.15) mV, BMSC Group +rAd.SERCA2a was (0.55 + 0.16) mV, which was significantly higher than that of the control group (0.23 + 0.1) mV (P 0.05), field potential BMSC group (104.5 + 25.43) ms and BMSC+rAd.SERCA2a group (107.67 + 24.01) ms, which were significantly longer than those of the control group (63 + 20.34) ms (P 0.05).BMSC+rAd.SERCA2a group was the shortest conduction time, and could significantly improve myocardial infarction surface ventricular muscle tissue. The homogeneity conduction of.4) group rAd.SERCA2a [(82.62 + 2.58)%], group BMSC [(80.24 + 4.15)%], BMSC+rAd.SERCA2a Group [(84.28 + 2.46)%] compared with shame Group [(70.49 + 3.27)%], compared with shame group (P0.05, n=6), and rAd.LacZ group [(71.34 + 2.42)%] had no statistical significance compared with shame group (P0.05, n=5). Body surface electrocardiogram can be found. Shame Group [(102.42 + 20.67) ms, n=7], QT interval in rAd.SERCA2a Group [(83.07 + 17.56) ms, n=6] and BMSC+rAd.SERCA2a Group [(81.20 + 5.64) ms, n=7] mean significantly decreased 18.9% and 20.7% (P0.05). In the group rAd.SERCA2a (121.25 + 18.64) ms BMSC group (106.12 + 20.76) ms and BMSC+rAd.SERCA2a group (106.35 + 19.51) ms (n=6) significantly longer than group shame [(60.45 + 19.12) ms, n=6] (P0.05)), the ventricular potential of ventricular myocardium in myocardial infarction area was significantly increased. .rAd.SERCA2a and BMSC+rAd.SERCA2a groups can inhibit the degeneration and necrosis of the infarcted myocardium in tissue morphology. Conclusion: 1) this study established a stable and reliable experimental method for the amplification of rAd.SERCA2a with the aid of HEK-293 cell culture. The recombinant adenovirus of the purified SERCA2a gene can be used directly. Animal experimental studies on cardiovascular diseases also have a certain reference value to the amplification and purification of recombinant adenovirus carrying other genes.2) BMSC is an effective gene therapy carrier.3.) adenovirus mediated SERCA2a gene overexpression can effectively enhance cardiac function and increase infarct heart beat in the short term. .4 SERCA2a gene, BMSC transplantation and SERCA2a gene modified BMSC transplantation can significantly improve cardiac function after myocardial infarction, but the overexpression of SERCA2a gene can reduce myocardial necrosis and delay ventricular remodeling, and the rAd.SERCA2a group can increase the heart more effectively. The potential of ventricular myocytes in the infarct area improves the homogeneity of ventricular myocardium and can prevent the occurrence of arrhythmia after myocardial infarction (.5) MEA technique is an ideal technique for detecting the electrophysiological rhythms and frequencies of cardiac tissue in the animal model of cardiovascular disease and conduction activity..MEA technique in the study of cardiac electrical activity in cell or gene therapy It has important application value.

【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R542.22

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