本文關鍵詞：超聲微泡介導反義磷酸受納蛋白基因轉染效應的研究　出處：《浙江大學》2009年博士論文　論文類型：學位論文

  更多相關文章： 超聲 微泡 毛細血管通透性 基因轉染 磷酸受納蛋白 肌漿網(wǎng)鈣ATP酶 超聲 微泡 基因轉染 心功能

【摘要】： 第一部分超聲微泡增加心肌血管通透性并提高基因轉染效率 研究背景: 近幾十年來,心臟病的診治方法有了長足的發(fā)展,但是心血管病仍然是人類死亡的主要原因之一�；蛑委熥鳛橐环N新的治療手段正處于探索階段。基因治療能否成功不僅取決于作用靶點的正確選擇,而且與載體以及傳輸系統(tǒng)是否奏效密切相關。直接心肌注射、心腔內注射或經(jīng)冠狀動脈傳輸基因都是常用的向心肌輸送外源性基因的方法,但是它們或者需要開胸,或者需要短暫心臟停搏,或者需要結扎主動脈和肺動脈,有一定的創(chuàng)傷性。 超聲波的“聲致孔”效應近年來被證實有利于基因的轉染。細胞膜在超聲的作用下可出現(xiàn)一過性的小孔,稱為聲致孔(sonoporation)效應,這些小孔可作為藥物或基因進入細胞內的通道,而該效應在微泡的參與下得到加強。內含氣體的微泡從靜脈注射提供了理想的空化核,使超聲介導的基因傳輸有了更廣闊的發(fā)展空間。 目的: 本研究用超聲破裂白蛋白氟碳氣體微泡,以伊文思藍為指示劑,研究增加心肌毛細血管通透性的優(yōu)化超聲參數(shù),并應用該參數(shù)介導報告基因在心肌的轉染,觀察超聲破裂微泡增強報告基因轉染效率的能力,為治療性基因在心肌的轉染提供實驗基礎。 方法: 構建含氟碳氣體的白蛋白微泡,從小鼠尾靜脈注射伊文思藍-微泡混合液,研究目前已知的影響微泡破裂的參數(shù)對伊文思藍滲出至心肌組織間隙的影響,包括超聲探頭頻率和機械指數(shù),選擇伊文思藍滲出最明顯的組合進行報告基因pAAV-LacZ在心肌的轉染。轉染后進行X-gal染色和β-半乳糖苷酶活性定量,評價該基因在心肌的轉染效果,并初步觀察對小鼠心臟功能的影響以及在其他臟器的轉染情況。 結果: 1.可引起伊文思藍滲出明顯增加的超聲參數(shù)組合為S3探頭,1.3 MHz,MI1.6,應用每8個心動周期觸發(fā)。聯(lián)合微泡注射,伊文思藍滲出可較單純注射伊文思藍組增加(251.59±16.4比30.87±4.4.26,p0.05),達8.15倍。組織切片觀察未見紅細胞滲出。 2.以該超聲參數(shù)設置聯(lián)合微泡注射進行的報告基因轉染顯示,注射第10天,X-gal染色顯示心肌細胞胞漿內有β-半乳糖苷酶表達,定量檢測較單純基因注射組明顯增加,達6.56倍,亦明顯高于基因注射加超聲但不含微泡組。第20天時心肌內不能檢測到β-半乳糖苷酶表達。 3.肝臟、肺、腦均未見β-半乳糖苷酶表達,僅腎臟腎小管上皮細胞內可見β-半乳糖苷酶表達,定量檢測顯示超聲破裂微泡組與對照組無明顯差別,提示為腎臟內源性β-半乳糖苷酶。 4.報告基因注射第10天、第20天超聲心動圖檢查未見小鼠心臟收縮功能改變。 結論: 1.超聲破裂微泡在合適的超聲參數(shù)下能明顯增強心肌毛細血管通透性�！　� 2.采用S3探頭,MI1.6,每8個心動周期觸發(fā)的方式在增加心肌毛細血管通透性的同時,不影響健康大鼠的心臟收縮功能,不引起紅細胞的滲出。 3.經(jīng)靜脈注射的方法通過靶向超聲破裂微泡能使報告基因在心臟成功轉染,且基因轉染具有器官特異性。 第二部分超聲微泡介導反義磷酸受納蛋白基因轉染效應的研究 研究背景: 磷酸受納蛋白(PLB)是心肌細胞肌漿網(wǎng)鈣ATP酶(SERCA_(2a))活性的最重要的調控蛋白。而PLB絲氨酸16(Ser16)殘基的磷酸化下調是SERCA_(2a)活性下降的重要原因。體內和體外的研究已證實,抑制PLB蛋白的表達,提高PLB的磷酸化水平,能提高SERCA_(2a)的活性,從而改善心臟的收縮和舒張功能。 反義PLB(asPLB)轉染心肌細胞能有效抑制細胞內PLB的表達,增強SERCA活性,因而改善心肌梗死大鼠的心臟收縮功能。但是外源性基因導入活體心肌的常用方法是直接心肌注射或心腔注射,這對于心力衰竭病人無疑有極大的風險。超聲破裂微泡進行靶向基因轉染作為一種無創(chuàng)的手段正處于研究階段。超聲微泡作為空化內核聚焦了超聲能量,降低了聲致孔效應的閾值,使細胞膜或毛細血管壁通透性增加,從而讓生物活性大分子物質能較容易地進入細胞或組織間隙,因而有利于外源性基因的成功轉染。 目的: 本研究以超聲破裂微泡介導asPLB質粒轉染急性心肌梗死小鼠,觀察該方法對急性心肌梗死小鼠心肌PLB、Ser16-PLB和SRECA蛋白水平以及SERCA活性和左室收縮功能的影響,為心力衰竭基因治療提供一項實驗依據(jù)。 方法: 構建含pAAV-asPLB質粒的氟碳氣體白蛋白微泡,以結扎冠狀動脈左前降支的方法制備急性心肌梗死(MI)小鼠模型。以編碼β-半乳糖苷酶的pAAV-LacZ為報告基因指示轉染是否成功。MI小鼠隨機分組為只注射生理鹽水的MI+生理鹽水組(MI+saline);只注射LacZ質粒的MI+LacZ組;注射LacZ質粒和進行超聲照射的MI+LacZ+US組;注射LacZ質粒-微泡混合液,同時進行超聲照射的MI+lacZ+MB+US組;只注射asPLB質粒的MI+asPLB組;注射asPLB質粒和超聲照射的MI+asPLB+US組;注射asPLB-微泡混合液,同時進行超聲照射的MI+asPLB+MB+US組。設立正常對照和假手術組。術后經(jīng)尾靜脈進行相應的注射和超聲照射。手術后三周超聲心動圖測定小鼠左室大小和左室短軸縮短率(FS)、左室射血分數(shù)(LVEF)。小鼠處死后,測定左室心肌組織PLB、Ser16-PLB和SERCA蛋白水平及SERCA活性。 結果: 1.各報告基因注射組,僅MI+LacZ+MB+US組心肌組織有β-半乳糖苷酶表達。肝、肺和腦組織均未見表達,腎小管上皮細胞內探及內源性β-半乳糖苷酶表達。 2.與假手術組比,各組MI小鼠心肌SERCA蛋白水平未明顯改變;PLB蛋白水平明顯升高,Ser16-PLB蛋白水平下降;左室內徑明顯增大,FS、LVEF明顯下降。除MI+asPLB+MB+US組SERCA活性與假手術組無明顯差別外,其他各組均下降。 3.與MI+saline組相比,MI+asPLB和MI+asPLB+US組心肌PLB、Ser16-PLB蛋白水平及SERCA蛋白水平和活性均無明顯差別。左室內徑及FS、LVEF無明顯改善。 4.與MI+saline相比,MI+asPLB+MB+US組左室心肌組織PLB蛋白水平明顯較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)明顯較MI+saline升高。SERCA蛋白水平?jīng)]有明顯改變。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。 結論: 1.單純超聲照射不能使靜脈注射的質粒在心肌組織有效表達。 2.超聲破裂微泡能增強外源性基因在心肌組織的轉染。經(jīng)靜脈注射質粒和微泡混合液聯(lián)合胸前區(qū)超聲照射可以達到在心肌靶向性轉染的效果。 3.通過超聲破裂微泡技術,pAAV-asPLB在心肌的轉染能部分改善MI小鼠心臟收縮功能,抑制心肌PLB的過表達,提高SERCA活性,增加PLB在Ser16位的磷酸化水平,對SERCA蛋白表達水平無顯著影響。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R541;R346

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相關期刊論文 前1條

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本文編號：1397632