超声微泡介导反义磷酸受纳蛋白基因转染效应的研究

发布时间：2018-01-08 15:17

本文关键词：超声微泡介导反义磷酸受纳蛋白基因转染效应的研究　出处：《浙江大学》2009年博士论文　论文类型：学位论文

【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,p0.05),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,p0.05),Ser16-PLB水平(0.8±0.25比0.46±0.18,p0.05)和SERCA活性(3.00±0.29比2.12±0.30,p0.05)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%,p0.05)、LVEF(48.2±5.18%比39.14±5.38%,p0.05)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。
[Abstract]:The first part of ultrasonic microbubble increases myocardial permeability and improves gene transfection efficiency
Research background:
In recent decades, has made the development of the diagnosis and treatment of heart disease, but cardiovascular disease is still one of the major causes of human death. Gene therapy is a new therapeutic means are at the exploratory stage. The correct choice of the success of gene therapy depends not only on the target, vector and delivery system work closely related. Direct myocardial injection and intracardiac injection or by coronary delivery of gene are exogenous gene to myocardial transport commonly used, but they need or need a thoracotomy, or cardiac arrest, or ligation of the aorta and pulmonary artery, is invasive.
The sound hole "effect of ultrasonic" in recent years have proved beneficial to transfection gene. The cell membrane can appear a hole under the effect of ultrasonic, called acoustic pore (sonoporation) effect, these holes can be used as a drug or gene into cells of the channel, and the effect was strengthened by microbubbles the participation of the micro bubble containing gas. From intravenous injection provides an ideal gene transfer nucleus, the ultrasound mediated have a broader space for development.
Objective:
This study used ultrasound microbubble rupture of perfluorocarbon, with Evans blue as the indicator of increased optimization of ultrasound parameters of myocardial capillary permeability, and the application of the parameter mediated reporter gene in myocardial transfection, ultrasound observation of rupture of microbubbles enhanced gene transfection efficiency report ability, to provide experimental basis for therapeutic gene in myocardium transfection.
Method:
Construction of albumin perfluorocarbon microbubbles, from the tail vein injection of Evans blue microbubble mixture, effects of parameters on the currently known effects of microbubble destruction of Evans blue extravasation to myocardial tissue space, including ultrasonic frequency and mechanical index, choose the combination of Yi Wen's blue exudation most obvious report gene pAAV-LacZ in the myocardial transfection. After transfection by X-gal staining and beta galactosidase activity of this gene in quantitative evaluation of myocardial transfection effect, and to observe the effects on cardiac function in mice and transfected in other organs.
Result:
1. can be caused by ultrasonic parameters of Evans blue extravasation significantly increased as the S3 probe, 1.3 MHz, MI1.6, application of each of the 8 cardiac cycle trigger. Microbubble injection of Evans blue extravasation can be compared with the injection of Evans Blue Group (251.59 + 16.4 to 30.87 + 4.4.26, P0.05), up to 8.15 times. No red blood cells exudation of tissue slices were observed.
2. combined with the ultrasonic microbubble injection parameters gene transfection report showed that injection for tenth days, X-gal staining showed that the expression of beta galactosidase with myocardial cell cytoplasm, quantitative detection than single gene injection group was significantly increased, up to 6.56 times, was also higher than that of gene injection but not containing microbubbles plus ultrasound the twentieth day group. Myocardium cannot detect beta galactosidase expression.
3., no expression of beta galactosidase was found in the liver, lung and brain. Only the expression of beta galactosidase in the renal tubular epithelial cells was observed. The quantitative detection showed that there was no significant difference between the ultrasonic ruptured microbubbles group and the control group, suggesting that the endogenous beta galactosidase in the kidney.
4. reports of gene injection for tenth days and twentieth days of echocardiography did not change the changes of cardiac contractile function in mice.
Conclusion:
1. ultrasound ruptured microbubbles can obviously enhance myocardial capillary permeability under appropriate ultrasonic parameters.
2., the use of S3 probe, MI1.6, every 8 cardiac cycle triggers the increase of myocardial capillary permeability, and does not affect the cardiac systolic function of healthy rats, and does not cause the exudation of red blood cells.
3. the transfection of the reporter gene in the heart can be successfully transfected through a targeted ultrasound ruptured microbubble, and the gene transfection has an organ specificity.
Study on the transfection effect of antisense phosphoric acid receptor gene in the second part of ultrasound microbubbles
Research background:
Phospholamban (PLB) is a myocardial sarcoplasmic reticulum Ca2 + ATP enzyme (SERCA_ (2a)) the activity of the most important regulatory proteins. PLB (Ser16) 16 serine residues phosphorylated by SERCA_ (2a) is an important reason for the decreased activity. In vivo and in vitro studies have confirmed that inhibition of expression PLB protein, increase the phosphorylation level of PLB can improve the SERCA_ (2a) activity, which can improve cardiac systolic and diastolic function.
Antisense PLB (asPLB) transfection of myocardial cells can effectively inhibit the expression of PLB in the cell, enhance the activity of SERCA, thereby improving cardiac contractile function in rats with myocardial infarction. But the common method of exogenous gene into the myocardium in vivo is direct myocardial injection or intracardiac injection, this will undoubtedly have a great risk for patients with heart failure. Ultrasonic rupture targeting gene transfection as a non-invasive tool is in the research stage. Microbubble ultrasound microbubble cavitation as the core focus ultrasound energy was caused by the sound hole effect threshold is reduced, the cell membrane or the capillary wall permeability increased, so that the bioactive macromolecules can easily enter cell or tissue clearance, which is conducive to the successful transfection of exogenous gene.
Objective:
In this study, ultrasonic rupture of microbubble mediated transfection of asPLB plasmid in mice with acute myocardial infarction, observe the method of acute myocardial infarction in Mice Myocardial PLB, effects of Ser16-PLB and SRECA protein level and activity of SERCA and the left ventricular systolic function, to provide an experimental basis for gene therapy of heart failure.
Method:
To construct pAAV-asPLB plasmid containing albumin perfluorocarbon microbubbles by ligation of the left anterior descending coronary artery preparation method of acute myocardial infarction (MI) mouse model. By encoding beta galactosidase pAAV-LacZ gene transfection indicated the success of.MI mice were randomly divided into MI+ normal saline group (saline injection MI+saline); MI+LacZ group were injected with LacZ plasmid; LacZ plasmid and MI+LacZ+US injection group were exposed to ultrasound; injection of LacZ plasmid microbubble mixture, MI+lacZ+MB+US group and ultrasound irradiation; MI+ group asPLB injection of asPLB plasmid; MI+asPLB+US group were injected with asPLB plasmid and ultrasound irradiation microbubble mixture injection; asPLB- at the same time, MI+asPLB+MB+US group of ultrasound irradiation. A normal control group and sham operation group. After tail vein injection and corresponding ultrasound irradiation. Three weeks after surgery by echocardiography in mice The left ventricular size and left ventricular short axis shortening rate (FS) and left ventricular ejection fraction (LVEF) were measured. The PLB, Ser16-PLB and SERCA protein levels and SERCA activity in the left ventricular myocardium were measured after the mice were executed.
Result:
1. in each gene injection group, only MI+LacZ+MB+US group had the expression of beta galactosidase. No expression was found in liver, lung and brain tissues. The expression of endogenous beta galactosidase was detected in renal tubular epithelial cells.
2. compared with sham operated group, no significant change in protein level of myocardial SERCA MI mice of each group; PLB protein levels were significantly increased, Ser16-PLB protein level decreased; left ventricular diameter increased significantly, FS and LVEF decreased significantly. There was no significant difference in group MI+asPLB+MB+US and SERCA activity in the sham operation group, the other groups were decreased.
3., compared with group MI+saline, there was no significant difference in myocardial PLB, Ser16-PLB protein level and SERCA protein level and activity between group MI+asPLB and MI+asPLB+US. Left ventricular diameter and FS and LVEF did not improve significantly.
4. compared with MI+saline, MI+asPLB+MB+US group of left ventricular myocardium PLB protein level was significantly lower than MI+saline (1.45 + 0.38 to 2.05 + 0.31, P0.05), Ser16-PLB (0.8 + 0.25 to 0.46 + 0.18, P0.05) and the activity of SERCA (3 + 0.29 to 2.12 + 0.30, P0.05) compared with MI+ saline increased not obviously change of.SERCA protein level. Left ventricular FS (19.64 + 2.59% to 16.04 + 2.29%, P0.05), LVEF (48.2 + 5.18% to 39.14 + 5.38%, P0.05).
Conclusion:
1. simple ultrasound irradiation can not effectively express the plasmids injected into the myocardium.
2., ultrasound disruption microbubbles can enhance the transfection of exogenous genes into myocardium. Intravenous injection of plasmid and microbubble mixture combined with ultrasonic irradiation in the chest area can achieve the effect of targeted transfection in myocardium.
3., by ultrasound breaking microbubble technology, pAAV-asPLB transfection can partially improve the cardiac systolic function of MI mice, inhibit the over expression of PLB, increase SERCA activity, increase the phosphorylation level of PLB at Ser16 level, and have no significant effect on SERCA protein expression level.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R541;R346

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