新型两亲分子作为小分子干扰核酸输递载体的研究
文献类型：学位论文
| 作者 | 孙丹丹 |
| 学位类别 | 硕士 |
| 答辩日期 | 2012-04-16 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 张欣 |
| 关键词 | 非病毒基因载体 RNA干扰 bola型两亲分子 |
| 其他题名 | Research on a new bolaamphiphile as a delivery vector applied in siRNA interference |
| 学位专业 | 化学工程 |
| 中文摘要 | 基因载体是基因治疗成功的关键所在。基于病毒性载体存在的安全性和免疫原性等问题，开发无毒、高效的非病毒基因治疗载体成为目前基因治疗领域研究的焦点和难点。本论文采用一种新型的不对称bola两亲性分子(16-[4-(2,5-二氨基-氨基戊酸)-苯氧基]-棕榈酸[2-(2,3,4,5,6-五羟基-氨基己酸)-乙基]-酰胺，简写为Orn-C16-G)作为siRNA基因载体，制备了不同比例的Orn-C16-G与siRNA的复合物(bolaplexes)。通过原子力显微镜观察不同重量比例的复合物的形貌和尺寸大小，发现游离的siRNA为伸展的长链结构；重量比<1时，复合物分布较宽，存在600-800 nm的团状结构；重量比≥1 (重量比=1:1, 2:1, 4:1)时，复合物呈现稳定的单分散结构，粒径在100-200 nm范围内。本文研究了不同重量比的bola分子/siRNA复合物在Hela细胞的内吞效果。实验发现当重量比为1:1时，复合物中的siRNA转染的荧光强度最高，说明在这个比例时，复合物中bola与siRNA已经形成稳定的复合物结构，继续增加Orn-C16-G的量，反而导致多余的Orn-C16-G富集在纳米颗粒表面，不利于细胞的内化。流式细胞实验结果也证明了这一点，当复合物重量比=1:1时，荧光标记的siRNA进入的细胞数最多，达到约80%。固定复合物重量比=1:1，利用CCK-8法考察了一系列bola浓度下的复合物的细胞毒性，结果表明复合物在所测定的浓度范围内的细胞存活率都高于90%，这一性能显著优于目前市售的转染试剂Lipofectamine2000TM。上述实验结果表明Orn-C16-G是一种具有良好应用前景的siRNA转运载体，这些研究为基于bola类两亲性分子非病毒基因载体的研究提供了重要的理论数据和实验基础。 |
| 英文摘要 | The key to successful gene delivery is efficient gene delivery vectors. Viral vectors can be pathogenic and immunogenic, which raise safety concerns, though they are highly efficient. Therefore, non-viral gene delivery vectors which are efficient and non-toxic are an attractive alternative. In the present work, a novel unsymmetrical bolaamphiphile (16-[4-(2,5-diamino-pentanoylamino)-phenoxy]-hexadecanoicacid[2-(2,3,4,5,6-pentahydroxy-hexanoylamino) -ethyl]-amide, short for Orn-C16-G) was used as a vector for siRNA. Several weight ratios of bola/siRNA complexes (bolaplexes) were prepared, and the morphology and size were observed with atomic force microscopy (AFM). The free siRNA was extended long chain structure; complexes had a wide polydispersity index when the weight ratio of bola/siRNA was less than 1; the complexes exhibited stable monodisperse, nanosize between 100-200nm, when the ratio was no less than 1, e.g. 1:1, 2:1 and 4:1. Cellular uptake of bolaplexes in different weight ratios of bola and siRNA was detected in HeLa cells by fluorescence microscope. The fluorescence intensity of bolaplexes was the highest when the weight ratio of bola/siRNA was equal to 1:1. It demonstrated that the bola and siRNA in the complexes had formed stable complex structures at this ratio, increasing the weight of Orn-C16-G would induce Orn-C16-G to assemble on the surface of nanoparticles and not in favor of cellular uptake. Cellular internalization of bolaplexes at different weight ratio of bola and siRNA was also determined using flow cytometry. The transfection efficiency reached 78.86%. CCK-8 kit was used to test the cytotoxicity of the bolaplexes at different concentrations of bola when the weight ratio was 1:1. The results indicated that the cell viability at the tested concentration range was higher than 90%, which was significantly superior than commercial transfection agent liposome Lipofectamine2000TM. In conclusion, these experimental results demonstrate that Orn-C16-G is a potential non-viral gene delivery vector, and these provide very important data to fabricate bolaamphiphiles as an efficient non-viral gene delivery vector. |
| 语种 | 中文 |
| 公开日期 | 2013-09-25 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1872]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 孙丹丹. 新型两亲分子作为小分子干扰核酸输递载体的研究[D]. 中国科学院研究生院. 2012. |
入库方式： OAI收割
来源：过程工程研究所
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