摘要
背景:非病毒递送系统是一种很有前景的基因或药物递送方法。聚乙烯亚胺 (PEI) 是一把双刃剑。它通过内吞作用将自身内化到细胞中并提高基因转移效率。然而,强正电荷也使 PEI 对细胞具有高度毒性。超声靶向微泡破坏(UTMD)是一种很有前途的基因和药物递送非病毒方法,但其效率仍有待提高。 目的:本研究的目的是探索一种将超声与非病毒基因传递相结合的系统,用于治疗宫颈癌 HeLa 细胞。 方法:本研究合成了一种阳离子超声造影剂(CUCA),验证了其理化性质、基因承载能力和细胞毒性。我们在前人研究的基础上,进一步优化了以下输血参数,包括超声参数、微泡浓度、质粒浓度、细胞密度等参数。本实验设计比较以下六组:(1)质粒组(P组),质粒15 μg; (2) PEI+质粒组(PEI+P组),1μl含10nmol氮的PEI和1μg含3nmol磷酸的DNA,PEI/DNA比等于氮/磷酸比7进行转染; (3)超声+质粒组(US+P),质粒15 μg; (4)超声+阳离子脂质体超声造影剂+质粒组(UTMD+P组),质粒15μg+阳离子脂质体超声造影剂5%; (5)超声+阳离子脂质体超声造影剂+PEI+质粒组(UTMD+PEI+P组),PEI/DNA比值等于氮磷比7转染,阳离子脂质体超声造影剂5%; (6)空白组,未处理,显微镜下观察对Hela细胞的影响,流式细胞仪检测细胞凋亡效率,CCK 8检测细胞活力。 结果:优化后的转染参数在一定程度上提高了超声联合C-UCA的转染效率,但其转染效率仍低于支链聚乙烯亚胺(bPEI)25 kDa。通过研究UTMD联合PEI介导的survivin miRNA对HeLa细胞凋亡的影响,我们发现单独使用PEI和超声联合CUCA均能将survivin miRNA转染细胞,有效诱导HeLa细胞凋亡。然而,两种方法之间的协同作用并不显着。 结论:相比之下,超声、C-UCA和PEI联合使用可能会显着降低UTMD和PEI的转染效率,具体机制有待进一步研究。
关键词: 超声、阳离子造影剂、基因治疗、PEI、肿瘤细胞凋亡、存活蛋白 miRNA。
Current Molecular Medicine
Title:Inducing Tumor Cell Apoptosis: Mediated Survivin miRNA by Ultrasound and Cationic Lipid Contrast Agent
Volume: 21 Issue: 9
关键词: 超声、阳离子造影剂、基因治疗、PEI、肿瘤细胞凋亡、存活蛋白 miRNA。
摘要:
Background: Non-viral delivery systems is a promising method for gene or drug delivery. Polyethyleneimine (PEI) is a double-edged sword. It internalizes itself into the cell through endocytosis and promotes gene transfer efficiency. However, the strong positive charge also makes PEI highly toxic to cells. Ultrasound-targeted microbubble destruction (UTMD) is a promising non-viral method for gene and drug delivery, but its efficiency still needs to be improved.
Objective: The aim of this study was to explore a system that combines ultrasound with non-viral gene delivery for the treatment of cervical cancer HeLa cells.
Methods: In this study, we synthesized a kind of cationic ultrasound contrast agent(CUCA) that the physical and chemical properties, gene carrying capacity and cytotoxicity were verified. On the basis of previous studies, we further optimized the following transfusion parameters including ultrasound parameters, microbubble concentration, plasmid concentration, cell density and other parameters. The experiment was designed to compare the following six groups: (1) Plasmid group (P group), plasmid 15 μg; (2) PEI + plasmid group (PEI + P group),1 μl of PEI containing 10 nmol nitrogen and 1 μg of DNA containing 3 nmol phosphate for a PEI/DNA ratio equal to a nitrogen/phosphate ratio of 7 for transfection; (3) Ultrasound + plasmid group (US + P), plasmid 15 μg; (4) Ultrasound + cationic liposomal ultrasound contrast agent + plasmid group (UTMD + P group), plasmid 15 μg and cationic liposomal ultrasound contrast agent 5%; (5) Ultrasound + cationic liposomal ultrasound contrast agent + PEI + plasmid group (UTMD + PEI + P group), PEI/DNA ratio equal to a nitrogen/phosphate ratio of 7 for transfection and cationic liposomal ultrasound contrast agent 5%; and (6) Blank group, no treatment), The influence on Hela cells was observed under microscope, the efficiency of apoptosis was measured by flow cytometry, and cell viability was tested in CCK 8.
Results: The optimized transfection parameters can improve the transfection efficiency of ultrasound combined with C-UCA to a certain extent, but its transfection efficiency is still lower than that of branched polyethyleneimine (bPEI) 25 kDa. By investigating the effect of HeLa cells apoptosis induced by UTMD in combination with PEI mediated survivin miRNA, we found that both PEI alone and ultrasound in combination with CUCA were able to transfect cells with survivin miRNA to effectively induce HeLa cell apoptosis. However, the synergistic effect between the two methods was not significant.
Conclusion: In contrast, the combined use of ultrasound, C-UCA and PEI may significantly reduce the transfection efficiency of UTMD and PEI, and the specific mechanism remains to be further studied.
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Cite this article as:
Inducing Tumor Cell Apoptosis: Mediated Survivin miRNA by Ultrasound and Cationic Lipid Contrast Agent, Current Molecular Medicine 2021; 21 (9) . https://dx.doi.org/10.2174/1566524020666200909164513
DOI https://dx.doi.org/10.2174/1566524020666200909164513 |
Print ISSN 1566-5240 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5666 |
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