發(fā)布時間：2018-06-19 14:31

  本文選題：超聲 + 微泡��； 參考：《重慶醫(yī)科大學》2007年碩士論文

【摘要】： 第一節(jié)超聲輻照微泡促進肝細胞基因轉(zhuǎn)染的參數(shù)優(yōu)化 目的:觀察超聲輻照微泡對正常肝細胞生長狀態(tài)的影響并優(yōu)化其促進肝細胞株L02基因轉(zhuǎn)染的參數(shù)。 方法:采用MTT比色法檢測單純超聲、單純微泡和超聲輻照微泡對正常肝細胞(L02細胞)生存率的影響及用熒光顯微鏡和流式細胞儀觀察和檢測超聲輻照微泡介導EGFP質(zhì)粒對L02細胞基因轉(zhuǎn)染的效率,并以此優(yōu)化聲照參數(shù)和微泡劑量。 結(jié)果:MTT比色法結(jié)果發(fā)現(xiàn):當固定聲強為0.5 W/cm~2,輻照時間為30 s、60 s和90 s時,各組細胞存活率分別為92.45%、85.78%和31.65%;采用不同濃度微泡對L02細胞作用24 h后,各實驗組細胞存活率與空白對照組細胞存活率比較無顯著性差異(P0.05);超聲+微泡(40μl)組L02細胞,光密度值下降,細胞存活率降低,與對照組比較差異有顯著性。超聲輻照微泡10μl、20μl、30μl各亞組的L02細胞生長狀態(tài)良好;熒光顯微鏡觀測發(fā)現(xiàn)在超聲輻照微泡組及脂質(zhì)體組均可見明亮的綠色熒光,對照組組未見熒光;流式細胞儀分析各組轉(zhuǎn)染效率顯示脂質(zhì)體組、超聲輻照微泡10μl微泡亞組、20μl微泡亞組、30μl微泡亞組、40μl微泡亞組轉(zhuǎn)染效率分別為18.30%、10.14%、14.65%、18.54%、10.06%,而對照組未見基因轉(zhuǎn)染。 結(jié)論:超聲頻率為1MHz、能量為0.5 W/cm~2輻照微泡(微泡濃度1.8×10~7個/ml)輻照時間為60秒可顯著促進EGFP基因在L02細胞的表達,并對L02細胞生長狀態(tài)無明顯影響。 第二節(jié)超聲輻照微泡增強脂質(zhì)體介導肝細胞基因轉(zhuǎn)染的實驗研究 目的:觀察超聲輻照微泡聯(lián)合脂質(zhì)體介導pIRES-EGFP/HGF質(zhì)粒對人正常肝細胞株L02的基因轉(zhuǎn)染效率。 方法:將培養(yǎng)的肝細胞分為5組,①單純對照組,②脂質(zhì)體轉(zhuǎn)染組,③超聲輻照脂質(zhì)體轉(zhuǎn)染組,④超聲輻照微泡轉(zhuǎn)染組,⑤超聲輻照微泡+脂質(zhì)體轉(zhuǎn)染組。采用熒光顯微鏡和流式細胞儀法觀察及檢測各組L02細胞pIRES-EGFP/HGF質(zhì)粒轉(zhuǎn)染的情況及效率;用MTT和ELISA法分別檢測各組L02細胞增殖率及上清液中HGF含量。 結(jié)果:流式細胞儀檢測結(jié)果顯示脂質(zhì)體組、超聲輻照脂質(zhì)體組、超聲輻照微泡組、超聲輻照微泡+脂質(zhì)體組轉(zhuǎn)染效率分別為18.32%、18.46%、18.47%、23.17%,對照組未見基因轉(zhuǎn)染。ELISA法結(jié)果顯示,超聲輻照微泡+脂質(zhì)體轉(zhuǎn)染組細胞上清液中HGF含量最高與其余各組比較差異有顯著性(P0.05);MTT比色法結(jié)果顯示對照組、脂質(zhì)體組、超聲輻照脂質(zhì)體組、超聲輻照微泡組、超聲輻照微泡+脂質(zhì)體組L02細胞增殖率分別為100%、117.79%、119.52%、118.44%、123.21%。 結(jié)論:超聲輻照微泡可明顯增強脂質(zhì)體介導pIRES-EGFP/HGF轉(zhuǎn)染人肝細胞株L02的效率,其效率明顯高于單純超聲輻照微泡組、超聲輻照脂質(zhì)體組及單純脂質(zhì)體組。
[Abstract]:Optimization of parameters of Ultrasound irradiated microbubbles in promoting Gene transfection of Hepatocytes objective: to observe the effect of Ultrasound irradiation on the growth of normal Hepatocytes and to optimize the effects of Ultrasound irradiation on the Promotion of Hepatic fineness Parameters of L02 gene transfection. Methods: MTT colorimetric assay was used to detect ultrasound. The effect of microbubbles and ultrasound on the survival rate of L02 cells and the efficiency of transfection of EGFP plasmid mediated by microbubbles on L02 cells were observed and detected by fluorescence microscope and flow cytometry. The sound radiation parameters and the dose of microbubbles were optimized. Results the cell survival rate of each group was 92.45% and 31.6555% respectively when the fixed sound intensity was 0.5 W / cm ~ 2 and the irradiation time was 30 s ~ 60 s and 90 s, respectively, and the cell survival rate was 92.45% and 31.6555%, respectively, after 24 h exposure to L02 cells with different concentrations of microbubbles. There was no significant difference in cell survival rate between the experimental group and the blank control group (P 0.05). The optical density of L02 cells decreased and the cell survival rate decreased in the ultrasound microbubble 40 渭 l group, which was significantly different from that in the control group. The growth status of L02 cells was good in 10 渭 l ~ 20 渭 l ~ (30 渭 l) groups, and the fluorescence microscope showed that bright green fluorescence could be observed in the ultrasound irradiated microbubbles group and liposome group, but no fluorescence was found in the control group. Flow cytometry analysis showed that the transfection efficiency of each group was the liposome group. The transfection efficiency of the 20 渭 l microbubble subgroup and 20 渭 l microbubble subgroup was 18.3010 渭 l microbubble subgroup and 40 渭 l microbubble subgroup respectively. The transfection efficiency of the 40 渭 l microbubble subgroup was 18.300.10.141.The transfection efficiency was 14.65% and 18.54% respectively, but no gene transfection was found in the control group. Conclusion: ultrasound frequency 1 MHz, energy 0.5 W / cm ~ 2 irradiation time of 60 seconds can significantly promote the expression of EGFP gene in L02 cells, and have no effect on the growth state of L02 cells. Experimental study on enhanced Liposome mediated Gene transfection of Hepatocytes by Ultrasound irradiation with microbubbles objective: to observe the effect of microbubbles combined with liposome mediated pIRES-EGFP / HGF plasmid on human normal liver Transfection efficiency of L02 gene. Methods: the cultured hepatocytes were divided into 5 groups: control group (control group), liposome transfection group (n = 5), microbubble transfection group (n = 5) and liposome transfection group (n = 5). The transfection efficiency of pIRES-EGFP / HGF plasmid in L02 cells was observed by fluorescence microscope and flow cytometry, and the proliferation rate and HGF content in supernatant of L02 cells were detected by MTT and Elisa, respectively. Results: the results of flow cytometry showed that the transfection efficiency of liposome group and ultrasound irradiated microbubble liposome group were 18.32 ~ 18.46 and 18.47 ~ 23.17, respectively. The content of HGF in the supernatant of ultrasound irradiated microbubble liposome transfection group was the highest compared with other groups. The results of MTT colorimetry showed that the control group, liposome group and ultrasound irradiated microbubble group had the highest HGF content. The proliferation rate of L02 cells in ultrasound irradiated microbubble liposome group was 100 and 117.79, and 119.52, 118.44 and 123.21, respectively. Conclusion: ultrasound irradiation can significantly enhance the efficiency of liposome-mediated pIRES-EGFP / HGF transfection into human liver cell line L02, and its efficiency is significantly higher than that of ultrasound irradiated microbubble group, ultrasound irradiated liposome group and pure liposome group.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R346

【參考文獻】

相關(guān)期刊論文 前8條

1 李曉東;超聲波空化效應的生物學機制[J];臨床超聲醫(yī)學雜志;2004年01期

2 李玲,王志剛;超聲微泡造影劑攜帶基因治療與超聲空化效應[J];臨床超聲醫(yī)學雜志;2005年03期

3 張麗梅,劉殿武;肝炎和肝纖維化基因治療的研究進展[J];臨床薈萃;2004年19期

4 ;Ultrasound/Microbubble Enhances Foreign Gene Expression in ECV304 Cells and Murine Myocardium[J];Acta Biochimica et Biophysica Sinica;2004年12期

5 程文,張青萍,貢雪灝,孔心涓;外周靜脈注入超聲造影劑介導p53基因治療大鼠肝癌的實驗研究[J];中國超聲醫(yī)學雜志;2003年08期

6 凌智瑜,王志剛,冉海濤,宮琳,黃晶,羅子國;超聲微泡造影劑對心肌組織毛細血管通透性的影響實驗研究[J];中國超聲醫(yī)學雜志;2004年05期

7 冉海濤,任紅,王志剛,唐海林,凌智瑜,羅子國;超聲波空化效應對體外培養(yǎng)細胞細胞膜作用的實驗研究[J];中華超聲影像學雜志;2003年08期

8 程文,張青萍,貢雪灝,孔心涓;超聲引導下瘤體內(nèi)注入超聲造影劑和p53基因治療大鼠肝癌的初步研究[J];中華超聲影像學雜志;2004年07期

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