本文選題：超聲微泡造影劑 + 重組腺相關(guān)病毒��； 參考：《重慶醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 背景:已有報道,超聲微泡造影劑能夠增強質(zhì)粒介導(dǎo)報告基因轉(zhuǎn)染體內(nèi)外視網(wǎng)膜神經(jīng)節(jié)細胞的效率。 目的:探討超聲微泡造影劑能否提高重組腺相關(guān)病毒(rAAV2)介導(dǎo)增強型綠色熒光蛋白(EGFP)基因在體內(nèi)轉(zhuǎn)染視網(wǎng)膜神經(jīng)節(jié)細胞(RGCs)的效率,并初步探討其安全性。 設(shè)計、時間及地點:基因形態(tài)學(xué)觀察實驗,于2008-03/09在重慶醫(yī)科大學(xué)超聲影像學(xué)研究所實驗室完成。 方法:75只SD大鼠隨機分為五組:A組(15只):玻璃體腔注射磷酸鹽緩沖液(PBS);B組(15只):玻璃體腔注射rAAV2-EGFP溶液;C組(15只):玻璃體腔注射rAAV2-EGFP溶液后立即用超聲輻照眼球;D組(15只):玻璃體腔注射rAAV2-EGFP和微泡造影劑的混懸液;E組(15只):玻璃體腔注射rAAV2-EGFP和微泡造影劑的混懸液后立即用超聲輻照眼球。玻璃體腔住射后第21天,用3%熒光金逆行標記RGCs。術(shù)后第28天,取出眼球,制作視網(wǎng)膜鋪片、視網(wǎng)膜冰凍切片及視網(wǎng)膜病理切片。 主要觀察指標:在激光共聚焦顯微鏡下觀察視網(wǎng)膜冰凍切片并計算EGFP基因在RGCs中表達的平均熒光密度;在激光共聚焦顯微鏡下觀察視網(wǎng)膜鋪片并計算EGFP基因在RGCs的轉(zhuǎn)染率及在RGCs表達的平均熒光密度;在視網(wǎng)膜鋪片上進行RGCs計數(shù)及觀察視網(wǎng)膜病理切片以判斷視網(wǎng)膜的損傷情況。 結(jié)果: 熒光金成功標記RGCs,在B、C、D、E四組均可觀察到RGCs中有EGFP表達。其中,B、C、D、E組RGCs轉(zhuǎn)染率分別是12.75±1.33%、15.78±0.31%、17.54±2.01%、20.10±0.74%,經(jīng)統(tǒng)計分析,差異有統(tǒng)計學(xué)意義(P0.05)。在視網(wǎng)膜冰凍切片及鋪片中計算各組平均熒光密度可知,E組平均熒光密度最強,均明顯高于B、C、D組,統(tǒng)計學(xué)上具有顯著差異性(P0.01)。A、B、C、D、E五組RGCs計數(shù)經(jīng)統(tǒng)計學(xué)分析,差異無統(tǒng)計學(xué)意義(P0.05)。B、C、D、E四組視網(wǎng)膜各層組織結(jié)構(gòu)與正常大鼠視網(wǎng)膜各層組織結(jié)構(gòu)無明顯差異。 結(jié)論:在一定條件下,超聲微泡造影劑能夠在體內(nèi)安全、有效地提高rAAV2介導(dǎo)EGFP基因轉(zhuǎn)染RGCs的效率。
[Abstract]:Background: it has been reported that ultrasound microbubble contrast agent can enhance the efficiency of plasmid mediated reporter gene transfection into retinal ganglion cells in vivo and in vitro. Aim: to investigate whether ultrasound microbubble contrast agent can improve the efficiency of recombinant adeno-associated virus (rAAV2) -mediated enhanced green fluorescent protein (EGFP) gene transfection into retinal ganglion cells (RGCs) and its safety. Design, time and place: gene morphological observation experiment, 2008-03 / 09, Laboratory of Ultrasonic Imaging Institute, Chongqing Medical University. Methods Seventy-five Sprague-Dawley rats were randomly divided into five groups: group A (n = 15): group B (n = 15) treated with phosphate buffer solution (PBS); group C (n = 15) treated with rAAV2-EGFP solution: group D (n = 15): group D was irradiated by ultrasound immediately after injection of rAAV2-EGFP solution into vitreous cavity. (15 rats): the suspension of rAAV2-EGFP and microbubble contrast agent was injected into vitreous cavity (n = 15): the suspension of rAAV2-EGFP and microbubble contrast agent was injected into vitreous cavity and the eyeball was irradiated with ultrasound immediately after injection. RGCs were retrograde labeled with 3% fluorescent gold on the 21st day after vitreous cavity irradiation. On the 28th day after operation, the eyeball was taken out, retinal slices were made, retinal frozen sections and retinal pathological sections were made. Main outcome measures: the frozen sections of retina were observed under laser confocal microscope and the average fluorescence density of EGFP gene expression in RGCs was calculated. The transfection efficiency of EGFP gene in RGCs and the average fluorescence density of EGFP gene expression in RGCs were calculated under laser confocal microscope, and the RGCs count and retinal pathological sections were observed to judge the damage of retina. Results: the expression of EGFP in RGCs was observed in four groups. The transfection efficiency of RGCs was 12.75 鹵1.33 and 15.78 鹵0.31 and 17.54 鹵2.01and 20.10 鹵0.74, respectively. The difference was statistically significant (P0.05). The mean fluorescence density of group E was higher than that of group B (P 0.01), and the mean fluorescence density of group E was significantly higher than that of group B (P 0.01). The count of RGCs in group C (P 0.01) was significantly higher than that in group C (P 0.01). The RGCs count in group E was significantly higher than that in group B (P 0.01). There was no significant difference between the four groups (P0.05). There was no significant difference in the tissue structure of each layer of retina between the four groups and the normal rats. Conclusion: under certain conditions, ultrasound microbubble contrast agent is safe in vivo and can effectively improve the efficiency of EGFP gene transfection into RGCs mediated by rAAV2.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R774.1

【引證文獻】

相關(guān)期刊論文 前1條

1 金利芳;杜聯(lián)芳;李凡;;超聲靶向破壞微泡介導(dǎo)腺相關(guān)病毒為載體的基因轉(zhuǎn)染[J];中國醫(yī)學(xué)影像技術(shù);2012年03期

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本文編號：2110420