本文選題：肌漿網(wǎng)鈣ATP酶 + 心肌梗塞　； 參考：《新疆醫(yī)科大學(xué)》2013年博士論文

【摘要】：目的：1)通過擴增和純化重組腺病毒攜帶肌漿網(wǎng)鈣ATP酶(rAd.SERCA2a)基因,獲得高效價的rAd.SERCA2a基因,可滿足基因轉(zhuǎn)染濃度和數(shù)量的需要,同時也為擴增和純化肌質(zhì)網(wǎng)Ca2+-ATP酶(SERCA2a)基因提供一種穩(wěn)定高效的實驗方法,也為心力衰竭(HF)的基因治療研究提供基礎(chǔ)。2)本研究是建立在急性心肌梗塞(AMI)動物模型基礎(chǔ)上,分別采用經(jīng)典的骨髓干細(xì)胞(BMSC)治療、SERCA2a基因治療以及SERCA2a基因修飾的BMSC移植(BMSC+SERCA2a)治療方法,運用組織病理技術(shù),無創(chuàng)心電技術(shù),超聲技術(shù),組織細(xì)胞電生理技術(shù),對比三種方法治療效果的差異性和優(yōu)越性,分別探討B(tài)MSC治療AMI、轉(zhuǎn)SERCA2a基因治療心肌梗塞后心力衰竭的有效性,以及兩者聯(lián)合治療的可行性,評價BMSC作為基因載體策略的可行性。本研究在改善心臟功能,糾正心梗后心力衰竭,防止心室重塑方面進(jìn)行研究,重點探討國、內(nèi)外研究都比較薄弱的環(huán)節(jié)即：轉(zhuǎn)SERCA2a基因治療/BMSC移植治療/BMSC+SERCA2a治療AMI后的心臟電一機械匹配和電生理特性的改變,導(dǎo)致/減少/防止心律失常的發(fā)生進(jìn)行評估,為以細(xì)胞移植為基礎(chǔ)的基因治療AMI提供臨床應(yīng)用基礎(chǔ)。方法：1)復(fù)蘇和傳代人胚腎細(xì)胞(HEK-293),用100μl1.9×1012pfu/ml rAd.SERCA2a感染HEK-293細(xì)胞,出現(xiàn)細(xì)胞病變效應(yīng)時收獲細(xì)胞,經(jīng)物理反復(fù)凍融方法及兩步氯化銫超速離心方法獲得純化的rAd.SERCA2a,紫外分光光度計比色法測定病毒DNA質(zhì)粒數(shù)。2)將急性心肌梗塞后心力衰竭動物模型制作成功的26只成年雄性SD大鼠隨機分為3組：假手術(shù)組(n=10),空病毒對照組(rAd.β-gal組,n=8)和SERCA2a基因轉(zhuǎn)染組(rAd.SERCA2a組,n=8)。假手術(shù)組僅開胸不結(jié)扎冠狀動脈,rAd.β-gal組和rAd.SERCA2a組分別進(jìn)行左冠狀動脈前降支結(jié)扎建立大鼠心肌梗塞后心力衰竭動物模型,同時分別將攜帶β-gal和SERCA2a基因的重組腺病毒導(dǎo)入衰竭心臟,術(shù)后2周超聲心電圖檢測心臟的舒張和收縮功能,心電圖監(jiān)測體表心電活動,微電極陣列(MEA)監(jiān)測離體心臟組織電活動情況。3)建立成年雄性SD大鼠AMI模型(n=24)。隨機分為3組：鹽水對照組(對照組,n=8),BMSC移植組(BMSC組,n=8),SERCA2a基因修飾的BMSC移植組(BMSC+rAd.SERCA2a組,n=8)。術(shù)后2周進(jìn)行心臟B超、體表心電圖及MEA技術(shù)評估心功能和心臟電活動變化。4)制作成年雄性SD大鼠AMI動物模型(n=38)。隨機分為5組：SERCA2a基因組(rAd.SERCA2a組,n=8),BMSC移植組(BMSC組,n=8),SERCA2a基因修飾的BMSC移植組(BMSC+rAd.SERCA2a組,n=8),鹽水對照組(shame組,n=7),腺病毒空載體組(rAd.LacZ組,n=7)。術(shù)后2周記錄體表心電圖以及運用心臟二維超聲評價心功能,HE染色評估心臟組織形態(tài)改變,運用MEA技術(shù)評估離體心臟組織電活動情況。結(jié)果：1)SERCA2a基因在HEK-293細(xì)胞成功呈綠色熒光表達(dá),純化的rAd.SERCA2a.GFP DNA質(zhì)粒數(shù)為1.3±0.58×1012pfu/ml, OD260/OD280比值為1.57±0.49(n=50)。2)rAd攜帶SERCA2a與β-gal基因均成功轉(zhuǎn)入大鼠衰竭心臟。Ad.SERCA2a組可改善心功能,與假手術(shù)組相比心室舒張末期容積與心室收縮末期容積有輕微的增加[(0.410±0.130) cm2對(0.39±0.02) cm2,(0.08±0.02) cm2對(0.06±0.01) cm2, P0.05]、左心室射血分?jǐn)?shù)[(82.3±4.59)%對(85.56±1.26)%,P0.05]和短軸縮短率[(46.6±2.32)%對(49.58±1.71)%,P0.05]無明顯改變。與假手術(shù)組相比,rAd.β-gal組體表心電圖QT間期延長[(111.02±7.42)ms對(94.7±1.55)ms,n=6,P0.05],室性早搏發(fā)生率達(dá)71.5%(5/7),而rAd.SERCA2a組QT間期縮短[(81.45±4.97)ms對(94.7±1.55) ms,n=6, P0.05],室性早搏發(fā)生率達(dá)14.3%(1/7)。MEA記錄可發(fā)現(xiàn)rAd.SERCA2a組心率與假手術(shù)組相比差異無統(tǒng)計學(xué)意義[(435±31)bpm對(442±22) bpm, n=6, P0.05],與rAd. β-gal組相比,rAd.SERCA2a組最大場電位[(0.82Q0.39)mV對(0.64±0.13)mV,n=6,P0.05]、最小場電位[(1.88±0.57)mV對(1.35±0.12) mV, n=6,P0.05]、場電位時限[(124.17±21.08)ms對(113.23±12.02) ms, n=6, P0.05]均延長；rAd.β-gal組梗死區(qū)與梗死對側(cè)區(qū)心肌組織場電位時限差異有統(tǒng)計學(xué)意義[(60.36±2.08)ms對(103.24±7.35) ms, n=5, P0.05],并且60通道記錄梗死區(qū)心肌組織場電位時程離散度大于rAd.SERCA2a組[(38.5ms±4.62) ms對(26.88±5.09) ms, n=5]; rAd.SERCA2a組傳導(dǎo)基本一致,使心肌梗塞面心室肌組織電活動呈均一性傳導(dǎo)。3)rAd.SERCA2a基因?qū)MSC的感染效率為80%～90%。BMSC組、BMSC+rAd.SERCA2a組大鼠左室射血分?jǐn)?shù)明顯優(yōu)于對照組(n=6,P0.05)。BMSC+rAd.SERCA2a組大鼠體表心電圖QT間期為(80.30±6.53)ms比對照組(105.31±21.89)ms少了23.8%(P0.05),對照組室性早搏較多。MEA記錄可發(fā)現(xiàn)梗死面心肌組織最大場電位BMSC組為(0.51±0.15)mV、BMSC+rAd.SERCA2a組為(0.55±0.16)mV,均明顯高于對照組的(0.23±0.1)mV(P均0.05),場電位時程BMSC組為(104.5±25.43)ms、BMSC+rAd.SERCA2a組為(107.67±24.01)ms,均顯著長于對照組的(63±20.34)ms(P均0.05)。BMSC+rAd.SERCA2a組傳導(dǎo)時間最短,且能顯著改善心梗面心室肌組織的均一性傳導(dǎo)。4)rAd.SERCA2a組[(82.62±2.58)%]、BMSC組[(80.24±4.15)%]、BMSC+rAd.SERCA2a組[(84.28±2.46)%]與shame組[(70.49±3.27)%]相比射血分?jǐn)?shù)均有明顯改善(P0.05,n=6),而rAd.LacZ組[(71.34±2.42)%]與shame組相比差異沒有統(tǒng)計學(xué)意義(P0.05,n=5)。體表心電圖可發(fā)現(xiàn)與shame組[(102.42±20.67)ms,n=7]相比,QT間期在rAd.SERCA2a組[(83.07±17.56)ms,n=6]和BMSC+rAd.SERCA2a組[(81.20±5.64)ms,n=7]均值明顯減少18.9%和20.7%(P0.05),在shame組和rAd.LacZ組也出現(xiàn)較多的室性早搏。MEA記錄可發(fā)現(xiàn)對照組離體心臟搏動頻率明顯減慢,并出現(xiàn)室性心律失常和房室傳導(dǎo)阻滯。離體心臟組織場電位時程(FPdur)在rAd.SERCA2a組(121.25±18.64) ms BMSC組(106.12±20.76)ms和BMSC+rAd.SERCA2a組(106.35±19.51)ms(n=6)顯著長于shame組[(60.45±19.12) ms, n=6](P0.05),其中rAd.SERCA2a組較顯著地增加心梗區(qū)心室肌場電位及減少心律失常發(fā)生。rAd.SERCA2a組和BMSC+rAd.SERCA2a組能夠從組織形態(tài)上阻止梗死心肌發(fā)生變性和壞死。結(jié)論：1)本研究建立了穩(wěn)定可靠的借助HEK-293細(xì)胞培養(yǎng)擴增rAd.SERCA2a的實驗方法,純化后的高效價的SERCA2a基因的重組腺病毒可直接用于心血管疾病的動物實驗研究,對重組腺病毒攜帶其他基因的擴增與提純方法也具有一定的參考價值。2)BMSC是一種有效的基因治療運載體。3)腺病毒載體介導(dǎo)的SERCA2a基因過度表達(dá)能夠在短期內(nèi)有效地增強心臟功能,增加梗死心臟的搏動頻律和顯著改善梗死心肌的電傳導(dǎo)和減少心律失常發(fā)生。4)SERCA2a基因、BMSC移植以及SERCA2a基因修飾BMSC移植均可以明顯改善心肌梗塞后心臟功能,但過表達(dá)SERCA2a基因可以減少心肌壞死和延緩心室重構(gòu),rAd.SERCA2a組可以更有效地增加心梗區(qū)心室肌場電位、改善心梗面心室的均一性傳導(dǎo),并能夠預(yù)防心梗后心律失常的發(fā)生。5)MEA技術(shù)是一項檢測心血管疾病動物模型心臟組織電生理節(jié)律和頻率以及傳導(dǎo)活動的理想技術(shù)。MEA技術(shù)在細(xì)胞或基因治療的心臟電活動研究方面具有重要的應(yīng)用價值。
[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.

【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R542.22

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