摘要: |
目的 研究脂氟显微泡及质粒浓度对pcDNA6.2-GW/EmGFP重组质粒转染效率及人肝癌HepG2细胞死亡率的影响,初步探索转染最佳条件及shRNA重组质粒对靶基因survivin的抑制效果。方法 将重组质粒加入各组,设置6个质粒浓度梯度组。向各组加入脂氟显微泡混悬液,设置6个微泡浓度梯度组。对照组不加入微泡和质粒。根据前期优化结果,采用治疗性超声(1.2W/cm2、占空比20%)辐照90s。48h后使用荧光显微镜及流式细胞仪检测细胞GFP表达效率。细胞死亡率通过台盼蓝染色计数获得。设立阳性质粒组P(+)和阴性质粒组P(-),western blot检测靶基因蛋白表达水平。 结果 当脂氟显微泡浓度小于等于 150μl/ml 时,转染率随微泡浓度增加而增加;当大于 150μl/ml 时,转染率却随微泡浓度增加而降低(p<0.05)。HepG2细胞死亡率随微泡浓度增高,呈上升趋势(p<0.05)。在一定范围(20μl/ml)内,增加质粒浓度可以提高转染效率(p<0.05);但继续增加质粒浓度对基因转染率无明显影响(p>0.05)。质粒浓度的改变对细胞死亡率无明显影响。Western blot结果表明, P(+)组survivin基因表达受到明显抑制。结论 超声联合微泡可以有效介导基因转染,当微泡浓度为 150μl/ml,质粒浓度为 20μl/ml时,基因转染率最高,细胞死亡率较低。阳性重组质粒P(+)可下调靶基因表达水平,为后续基因转染的相关研究奠定基础。 |
关键词: 微泡 声孔效应 肝癌 转染 |
DOI: |
投稿时间:2016-03-14修订日期:2016-03-14 |
基金项目:国家自然科学基金项目(面上项目,重点项目,重大项目)(NO.81350919); |
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Optimization of microbubble and plasmid concentration for gene transfection |
heying,zhuo zhong xiong |
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Abstract: |
Objective: To investigate the optimal microbubble concentration and pcDNA6.2-GW/EmGFP recombinant plasmid dosage for gene delivery, and determine the suppressive effect of the shRNA recombinant plasmid on the target gene. Methods Recombinant plasmid and microbubble were added in different groups. The microbubble and plasmid concentrations were varied within a range from 50μl/ml, 5μl/ml to 300μl/ml, 30μl/ml, respectively. The control group did not add any microbubble or plasmid. According to our previous research, therapeutic ultrasound (1.2 W/cm2, 20% duty cycle) was chose to irradiate each group 90s. 48 hours after transfection,the expression of GFP was detected by fluorescence microscopy and flow cytometry. The cell mortality was observed by trypan blue exclusion. Results GFP expression differed from each group. When the microbubble concentration was no more than 150μl/ml, the transfection rate increased as the increasing of microbubble concentration (p<0.05). However, if microbubble concentration exceeded 150μl/ml, the transfection rate decreased (p<0.05). The trend of cell mortality was deceased as microbubble concentration increased (p<0.05). Increasing plasmid dosage can get higher transfection rate in a certain range (20μl/ml), but continue increasing did not bring obviously benefit (p<0.05). Plasmid concentration had no significant correlation with cell mortality (p>0.05). Western blot result showed that target gene survivin was silenced, the expression of which was suppressed. Conclusion Ultrasound combined with microbubble can be used for effective gene delivery. The system concentration of 150μl/ml microbubble and 20μl/ml plasmid is the optimal condition for HepG2 transfecion. |
Key words: Microbubbles Sonoporation hepatocellular carcinoma transfection |