【作者】 牟芸；

【导师】 胡申江；

【作者基本信息】 浙江大学 ， 内科学， 2009， 博士

【摘要】 第一部分超声微泡增加心肌血管通透性并提高基因转染效率研究背景:近几十年来,心脏病的诊治方法有了长足的发展,但是心血管病仍然是人类死亡的主要原因之一。基因治疗作为一种新的治疗手段正处于探索阶段。基因治疗能否成功不仅取决于作用靶点的正确选择,而且与载体以及传输系统是否奏效密切相关。直接心肌注射、心腔内注射或经冠状动脉传输基因都是常用的向心肌输送外源性基因的方法,但是它们或者需要开胸,或者需要短暂心脏停搏,或者需要结扎主动脉和肺动脉,有一定的创伤性。超声波的“声致孔”效应近年来被证实有利于基因的转染。细胞膜在超声的作用下可出现一过性的小孔,称为声致孔（sonoporation）效应,这些小孔可作为药物或基因进入细胞内的通道,而该效应在微泡的参与下得到加强。内含气体的微泡从静脉注射提供了理想的空化核,使超声介导的基因传输有了更广阔的发展空间。目的:本研究用超声破裂白蛋白氟碳气体微泡,以伊文思蓝为指示剂,研究增加心肌毛细血管通透性的优化超声参数,并应用该参数介导报告基因在心肌的转染,观察超声破裂微泡增强报告基因转染效率的能力,为治疗性基因在心肌的转染提供实验基础。方法:构建含氟碳气体的白蛋白微泡,从小鼠尾静脉注射伊文思蓝-微泡混合液,研究目前已知的影响微泡破裂的参数对伊文思蓝渗出至心肌组织间隙的影响,包括超声探头频率和机械指数,选择伊文思蓝渗出最明显的组合进行报告基因pAAV-LacZ在心肌的转染。转染后进行X-gal染色和β-半乳糖苷酶活性定量,评价该基因在心肌的转染效果,并初步观察对小鼠心脏功能的影响以及在其他脏器的转染情况。结果:1.可引起伊文思蓝渗出明显增加的超声参数组合为S3探头,1.3 MHz,MI1.6,应用每8个心动周期触发。联合微泡注射,伊文思蓝渗出可较单纯注射伊文思蓝组增加（251.59±16.4比30.87±4.4.26,p<0.05）,达8.15倍。组织切片观察未见红细胞渗出。2.以该超声参数设置联合微泡注射进行的报告基因转染显示,注射第10天,X-gal染色显示心肌细胞胞浆内有β-半乳糖苷酶表达,定量检测较单纯基因注射组明显增加,达6.56倍,亦明显高于基因注射加超声但不含微泡组。第20天时心肌内不能检测到β-半乳糖苷酶表达。3.肝脏、肺、脑均未见β-半乳糖苷酶表达,仅肾脏肾小管上皮细胞内可见β-半乳糖苷酶表达,定量检测显示超声破裂微泡组与对照组无明显差别,提示为肾脏内源性β-半乳糖苷酶。4.报告基因注射第10天、第20天超声心动图检查未见小鼠心脏收缩功能改变。结论:1.超声破裂微泡在合适的超声参数下能明显增强心肌毛细血管通透性。　　2.采用S3探头,MI1.6,每8个心动周期触发的方式在增加心肌毛细血管通透性的同时,不影响健康大鼠的心脏收缩功能,不引起红细胞的渗出。3.经静脉注射的方法通过靶向超声破裂微泡能使报告基因在心脏成功转染,且基因转染具有器官特异性。第二部分超声微泡介导反义磷酸受纳蛋白基因转染效应的研究研究背景:磷酸受纳蛋白（PLB）是心肌细胞肌浆网钙ATP酶(SERCA_2a)活性的最重要的调控蛋白。而PLB丝氨酸16（Ser16）残基的磷酸化下调是SERCA_2a活性下降的重要原因。体内和体外的研究已证实,抑制PLB蛋白的表达,提高PLB的磷酸化水平,能提高SERCA_2a的活性,从而改善心脏的收缩和舒张功能。反义PLB（asPLB）转染心肌细胞能有效抑制细胞内PLB的表达,增强SERCA活性,因而改善心肌梗死大鼠的心脏收缩功能。但是外源性基因导入活体心肌的常用方法是直接心肌注射或心腔注射,这对于心力衰竭病人无疑有极大的风险。超声破裂微泡进行靶向基因转染作为一种无创的手段正处于研究阶段。超声微泡作为空化内核聚焦了超声能量,降低了声致孔效应的阈值,使细胞膜或毛细血管壁通透性增加,从而让生物活性大分子物质能较容易地进入细胞或组织间隙,因而有利于外源性基因的成功转染。目的:本研究以超声破裂微泡介导asPLB质粒转染急性心肌梗死小鼠,观察该方法对急性心肌梗死小鼠心肌PLB、Ser16-PLB和SRECA蛋白水平以及SERCA活性和左室收缩功能的影响,为心力衰竭基因治疗提供一项实验依据。方法:构建含pAAV-asPLB质粒的氟碳气体白蛋白微泡,以结扎冠状动脉左前降支的方法制备急性心肌梗死（MI）小鼠模型。以编码β-半乳糖苷酶的pAAV-LacZ为报告基因指示转染是否成功。MI小鼠随机分组为只注射生理盐水的MI+生理盐水组（MI+saline）;只注射LacZ质粒的MI+LacZ组;注射LacZ质粒和进行超声照射的MI+LacZ+US组;注射LacZ质粒-微泡混合液,同时进行超声照射的MI+lacZ+MB+US组;只注射asPLB质粒的MI+asPLB组;注射asPLB质粒和超声照射的MI+asPLB+US组;注射asPLB-微泡混合液,同时进行超声照射的MI+asPLB+MB+US组。设立正常对照和假手术组。术后经尾静脉进行相应的注射和超声照射。手术后三周超声心动图测定小鼠左室大小和左室短轴缩短率（FS）、左室射血分数（LVEF）。小鼠处死后,测定左室心肌组织PLB、Ser16-PLB和SERCA蛋白水平及SERCA活性。结果:1.各报告基因注射组,仅MI+LacZ+MB+US组心肌组织有β-半乳糖苷酶表达。肝、肺和脑组织均未见表达,肾小管上皮细胞内探及内源性β-半乳糖苷酶表达。2.与假手术组比,各组MI小鼠心肌SERCA蛋白水平未明显改变;PLB蛋白水平明显升高,Ser16-PLB蛋白水平下降;左室内径明显增大,FS、LVEF明显下降。除MI+asPLB+MB+US组SERCA活性与假手术组无明显差别外,其他各组均下降。3.与MI+saline组相比,MI+asPLB和MI+asPLB+US组心肌PLB、Ser16-PLB蛋白水平及SERCA蛋白水平和活性均无明显差别。左室内径及FS、LVEF无明显改善。4.与MI+saline相比,MI+asPLB+MB+US组左室心肌组织PLB蛋白水平明显较MI+saline降低（1.45±0.38比2.05±0.31,p<0.05）,Ser16-PLB水平（0.8±0.25比0.46±0.18,p<0.05）和SERCA活性（3.00±0.29比2.12±0.30,p<0.05）明显较MI+saline升高。SERCA蛋白水平没有明显改变。左室FS （19.64±2.59%比16.04±2.29%,p<0.05）、LVEF（48.2±5.18%比39.14±5.38%,p<0.05）上升。结论:1.单纯超声照射不能使静脉注射的质粒在心肌组织有效表达。2.超声破裂微泡能增强外源性基因在心肌组织的转染。经静脉注射质粒和微泡混合液联合胸前区超声照射可以达到在心肌靶向性转染的效果。3.通过超声破裂微泡技术,pAAV-asPLB在心肌的转染能部分改善MI小鼠心脏收缩功能,抑制心肌PLB的过表达,提高SERCA活性,增加PLB在Ser16位的磷酸化水平,对SERCA蛋白表达水平无显著影响。更多还原

【Abstract】 Part One Increase of capillary permeability and enhancement of naked plasmid DNA transfection in myocardium using ultrasound-mediated microbubble destruction in miceBackground:Diagnostic methods and therapeutic means to heart diseases have great improvement in recent years. But it still remains the leading cause of death across all populations. Cardiac gene therapy has already been investigated in experimental and some clinical studies. The success of gene therapy was determined by the effect of therapeutic target genes, efficiency of vector and delivery system. Direct intramyocardium injection, left ventricular cavity injection or intracoronary perfusion was common used. Some of them need surgery to open chest wall, or induce cardiac arrest, or clamp the aorta and pulmonary artery for a brief period of time. They are limited by procedure related risks.Sonoporation was defined as creation of transient, non-lethal holes in the plasma membrane with the assistance of ultrasound. It was reported extracellular molecules are able to diffuse through these holes thus facilitate gene transfer. Intravenous injection of microbubble act as cavitation nuclei enhances sonoporation effect and aid in a wide range of ultrasound-mediated drug delivery applications.Objective:This study was taken to optimize ultrasound parameters of increase of capillary permeability in myocardium by ultrasound-mediated destruction of albumin perfluorocarbon microbubbles via systemic injection of Evans blue dye. We also extended the method for plasmid deoxyribonucleic acid （DNA） transfection.Methods:We constructed albumin perfluorocarbon microbubbles. Evans blue was injected intravenously into mice via tail vein. We evaluated the effects of ultrasound parameters known to influent microbubble destruction, including ultrasound probe, ultrasound frequency and ultrasound mechanical index, on Evans blue extraction. The parameters caused greatest extent of Evans blue extraction was used to perform plasmid DNA transfection. X-gal staining andβ-galactosidase quantification were made to detect gene expression. In addition, mice left ventricular systolic function evidenced by echocardiography and gene expression in liver, lung and kidney were evaluated.Results:1. Optimal ultrasound parameter of this instruction was S3 transduce, 1.3MHz and mechanical index 1.6, with electrocardiogram triggering. Evans blue dye extraction reached utmost extent in this condition combined with microbubbles compared with dye injection without ultrasound and microbubbles （251.59±16.4 vs 30.87±4.26, p<0.05）. There was no red blood cells effusion in tissue sections.2. Expression ofβ-galactosidase was found in heart in the optimal parameter 10 days later, and the activity was markedly increased compared with plasmid injection olne or combined with ultrasound olne. Expression ofβ-galactosidase was not found 20 days later. 3. Expression ofβ-galactosidase was not found in liver, lung and brain by X-gal staining, but found in tubular epithelial cells of kidney in each group, including the control group without plasmid injection, indicated endogenousβ-galactosidase activity in kidney .4. Mice left ventricular systolic function was not significantly altered 10 days and 20 days after insonation and injection of plasmid DNA.Conclusion:1. Ultrasound destruction of microbubbles can increase capillary permeability significantly in myocardium under optimal parameter.2. Although capillary permeability increased, mice were secure from normal left ventricular systolic function and no red blood cells effusion.3. Ultrasound mediated-microbubble destruction enhanced reporter gene transfection to myocardium by injection of plasmid-microbubble mixture with simultaneous sonication. Targeted transfection maybe achieved by sonication through chest wall. Part Two Effects of asPLB gene transfection using ultrasound-mediated microbubble destructionBackground:Phospholamban （PLB） is a critical regulator of the cardiac sarcoplasmic reticulum Ca²⁺ ATPase (SERCA_2a) activity and cardiac contractility. Dephosphorylated PLB inhibits SERCA_2a activity, whereas phosphorylated PLB dissociates from SERCA_2a and leads to enhanced contractibility. PLB can be phosphorylated at the serine16 residue by cAMP-dependent protein kinase and at the threonine17 residue via CaMKⅡ. It has been shown that reduced serine16 phosphorylation of PLB （Serl6-PLB） in the failing rat myocardium is a major contributor to decreased SERCA_2a activity. In vitro and in vivo studies have demonstrated that inhibition of PLB expression, increase the phosphorylated PLB in myocardium can enhance SERCA_2a activity and then restore left ventricular systolic function in failing heart.Transfection of antisense PLB （asPLB） to myocardium of myocardial infarction rats can inhibit up-expression of PLB, and improve the cardiac function. But in vivo transfection of an exotic gene to heart usually requires surgical procedure or catheter-based endomyocardial approaches. They are invasive and expensive. Recently, ultrasound-mediated microbubble destruction has been proposed as a new technique for site-specific gene delivery as a noninvasive approach. Microbubbles act as the cavitation nuclei to focus ultrasound energy by lowering the threshold of sonoporation, which means transient ultrasound-induced increase in permeability of cell membrane or the capillary wall.Objective:We design to estimate the effect of gene transfer of pAAV-antisense phospholamban （pAAV-asPLB）, using ultrasound mediated microbubble destruction, on the left ventricular function, PLB and Serl6-PLB protein expression, cardiac sarcoplasmic reticulum Ca²⁺ ATPase (SERCA_2a) protein level and activity in myocardial infarction （MI） mice.Methods:MI mice were generated by ligating the left anterior descending coronary artery. Microbubbles were prepared by sonicated perfluorocarbon gas with dextrose and albumin. pAAV-LacZ was used as a reporter gene to determine the efficiency and localization of transfection. A mixture of pAAV-asPLB plasmid and microbubbles was injected via tail vein while the heart was simultaneously exposed to ultrasound via transthoracic insonation. Mice were divided into 9 groups as follows: normal control, sham-operation, MI+saline, MI+LacZ, MI+LacZ+US, MI+LacZ+MB+US, MI+asPLB, MI+asPLB+US, MI+asPLB+MB+US. Three weeks later, left ventricular ejection fraction （LVEF） and fraction shortening （FS） were measured by echocardiography. PLB, Ser16-PLB, SERCA protein level and SERCA activity were examined.Results:1. Expression ofβ-galactosidase was found in heart in MI+LacZ+MB+US group, not found in liver, lung and brain in each LacZ group, but found in tubular epithelial cells of kidney in each LacZ group and Ml+saline group, indicated endogenousβ-galactosidase activity in kidney .2. Compared with sham-operation, SERCA protein level was not significantly altered in MI mice; but PLB protein level increased significantly, Serl6-PLB decreased significantly; left ventricular diameter enlarged, FS and LVEF markedly depressed in MI groups. SERCA activity was decreased significantly in MI groups except MI+asPLB+MB+US.3. Compared with Ml+saline, PLB, Serl6-PLB, SERCA protein level and SERCA activity were not changed markedly in MI+asPLB, MI+asPLB+US. No significant improvement in left ventricular diameter, FS and LVEF in these two groups.4. LVEF （48.2±5.18% vs 39.1±5.38%, p<0.05）, FS （19.6±2.59% vs 16.0±2.29%, p<0.05）, SERCA activity （3.00±0.29 vs 2.12±0.30, p<0.05） and Serl6-PLB protein level （0.8±0.25 vs 0.46±0.18, p<0.05） were increased in MI+asPLB+MB+US group while PLB protein level （1.45±0.38 vs 2.05±0.31, p<0.05） was decreased compared with MI+saline. SERCA protein level was not statistical difference.Conclutions:1. Ultrasound alone can not achieve effective gene transfer in myocardium by systemic injection of plasmid.2. Ultrasound-mediated microbubble destruction can enhance exotic gene transfer to myocardium. Targeted transfection maybe acquired by systemic injection of plasmid-microbubble mixture with simultaneous sonication through chest wall.3. asPLB gene transfection can be achieved by ultrasound-mediated microbubble destruction. Effective transfection can partly restore heart function in MI mice, which inhibited myocardial PLB protein expression, restored phosphorylation of PLB on Ser16 site, and restored myocardial SERCA activity. It had no effects on myocardium SERCA protein level.更多还原