超声辐照微泡增强脂质体介导肝细胞基因转染的实验研究

发布时间：2018-06-19 14:31

本文选题：超声 + 微泡　；参考：《重庆医科大学》2007年硕士论文

【摘要】： 第一节超声辐照微泡促进肝细胞基因转染的参数优化目的:观察超声辐照微泡对正常肝细胞生长状态的影响并优化其促进肝细胞株L02基因转染的参数。方法:采用MTT比色法检测单纯超声、单纯微泡和超声辐照微泡对正常肝细胞(L02细胞)生存率的影响及用荧光显微镜和流式细胞仪观察和检测超声辐照微泡介导EGFP质粒对L02细胞基因转染的效率,并以此优化声照参数和微泡剂量。结果:MTT比色法结果发现:当固定声强为0.5 W/cm~2,辐照时间为30 s、60 s和90 s时,各组细胞存活率分别为92.45%、85.78%和31.65%;采用不同浓度微泡对L02细胞作用24 h后,各实验组细胞存活率与空白对照组细胞存活率比较无显著性差异(P0.05);超声+微泡(40μl)组L02细胞,光密度值下降,细胞存活率降低,与对照组比较差异有显著性。超声辐照微泡10μl、20μl、30μl各亚组的L02细胞生长状态良好;荧光显微镜观测发现在超声辐照微泡组及脂质体组均可见明亮的绿色荧光,对照组组未见荧光;流式细胞仪分析各组转染效率显示脂质体组、超声辐照微泡10μl微泡亚组、20μl微泡亚组、30μl微泡亚组、40μl微泡亚组转染效率分别为18.30%、10.14%、14.65%、18.54%、10.06%,而对照组未见基因转染。结论:超声频率为1MHz、能量为0.5 W/cm~2辐照微泡(微泡浓度1.8×10~7个/ml)辐照时间为60秒可显著促进EGFP基因在L02细胞的表达,并对L02细胞生长状态无明显影响。第二节超声辐照微泡增强脂质体介导肝细胞基因转染的实验研究目的:观察超声辐照微泡联合脂质体介导pIRES-EGFP/HGF质粒对人正常肝细胞株L02的基因转染效率。方法:将培养的肝细胞分为5组,①单纯对照组,②脂质体转染组,③超声辐照脂质体转染组,④超声辐照微泡转染组,⑤超声辐照微泡+脂质体转染组。采用荧光显微镜和流式细胞仪法观察及检测各组L02细胞pIRES-EGFP/HGF质粒转染的情况及效率;用MTT和ELISA法分别检测各组L02细胞增殖率及上清液中HGF含量。结果:流式细胞仪检测结果显示脂质体组、超声辐照脂质体组、超声辐照微泡组、超声辐照微泡+脂质体组转染效率分别为18.32%、18.46%、18.47%、23.17%,对照组未见基因转染。ELISA法结果显示,超声辐照微泡+脂质体转染组细胞上清液中HGF含量最高与其余各组比较差异有显著性(P0.05);MTT比色法结果显示对照组、脂质体组、超声辐照脂质体组、超声辐照微泡组、超声辐照微泡+脂质体组L02细胞增殖率分别为100%、117.79%、119.52%、118.44%、123.21%。结论:超声辐照微泡可明显增强脂质体介导pIRES-EGFP/HGF转染人肝细胞株L02的效率,其效率明显高于单纯超声辐照微泡组、超声辐照脂质体组及单纯脂质体组。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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