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新型负载海藻糖穿膜肽的设计
Alternative TitleDesign of Cell Penetrating Peptides for Intracellular Delivery of Trehalose into Mammalian Cells
李春龙
Subtype硕士
Thesis Advisor徐霞
2013-05-01
Degree Grantor中国科学院研究生院
Degree Discipline生物工程
Keyword穿膜肽   海藻糖   哺乳细胞   冷冻干燥
Abstract哺乳细胞的冻干制品在保存、运输和临床应用具有广阔的应用前景。海藻糖作为一种重要的细胞冻干保护剂,其本身不具有膜透过性,是海藻糖未能广泛应用于哺乳细胞的保存的原因。本研究以将海藻糖导入哺乳细胞为目的,运用计算机模拟的方法设计穿膜肽载体KRKRWHW。对设计的新型的穿膜肽进行合成并提纯,考察其细胞毒性和穿膜效率,并对其载运海藻糖的能力进行初步的探究。主要的结论如下:(1)利用Autodock分子对接软件,设计筛选出对膜上受体—肝素具有高亲和力的肽段RKKK、KRKR、RRKK。利用GROMACS分子动力学模拟软件,在水环境模拟条件下进一步的验证与分析,确定序列RKKK、KRKR、RRKK作为设计肽的肝素作用端的候选序列。(2)利用Autodock分子对接软件,设计筛选出对海藻糖具有高亲和力的肽段WWH、WHH、HHW、WHW。进一步利用GROMACS分子动力学模拟软件验证与分析,确定WWH、WHH、WHW作为海藻糖作用端的侯选序列。(3)根据肽的肝素作用端和海藻糖作用端,组合设计含有7个氨基酸残基的穿膜肽,利用GROMACS考察穿膜肽同海藻糖和肝素在水环境下的相互作用,获得最优的穿膜肽序列为KRKRWHW。(4)对所设计的穿膜肽KRKRWHW,进行合成与提纯,并进行质谱鉴定。得到产物含有目的肽96.3%,用于进一步的实验验证。(5)对穿膜肽的穿膜效率和细胞毒性进行了研究。对穿膜肽KRKRWHW的碳端进行FITC标记,对穿膜肽的穿膜作用进行了荧光分析,当荧光肽浓度为0.1mM时,孵育1h时,基本所有的细胞都已经有明显的荧光现象。对穿膜肽KRKRWHW对细胞的毒性进行了鉴定,穿膜肽高浓度下(5mM)孵育4h时,细胞的活率依然在90%以上。(6)在含有1mM KRKRWHW和8mM海藻糖的培养基条件下培养细胞2h,可以使细胞内海藻糖浓度达到25 mM左右,同时细胞活率同空白相比没有大的变化。设计的穿膜肽KRKRWHW在具有较好的穿膜效率的同时,可以高效的携带海藻糖进入细胞,为海藻糖导入哺乳细胞提供了新策略。
Other AbstractStabilization of mammalian cells in desiccated state can significantly simplify the storage and transportation and provide enormous benefits for clinical applications. Introduction of impermeable disaccharide, trehalose, into mammalian cells is an important step for mammalian cells to improve desiccation tolerance. In this study, a novel cell penetrating peptide (CPP), KRKRWHW, with low toxicity and high efficiency of penetrating into mammalian cells was developed based on the docking and molecular dynamics (MD) simulations. We evaluated the cytotoxicity of the designed CPP to mouse embryonic fibroblasts (MEFs) and further examined the efficiency of the designed CPP entering into living MEFs. The capacity of intracellular trehalose loading using the designed CPP was also determined. The main conclusions are as the following:(1) Based on the docking results, the peptides of RKKK, KRKR and RRKK were chosen due to their strong interactions with heparin sulfate. We further evaluated the interactions between the peptides and heparin using the GROMACS in the water environment at room temperature at 1 atm. The results revealed that the peptides of RKKK, KRKR and RRKK had great potential for cell penetration. (2) The peptides of WWH, WHH, HHW and WHW were selected as the candidates for coupling with trehalose through hydrogen bond and π-π bond based on the docking and MD results. (3) The sequence of KRKRWHW was selected as the potential CPP due to the low total free energy and stable conformation in the system composed of trehalose-peptide-heparin. (4) The designed peptide of KRKRWHW was synthesized, purified and identified. The purity of the products was 96.3%.(5) The translocation efficiency of KRKRWHW was determined using the FITC-labeled peptide. The peptide of KRKRWHW had capacity of cell penetration even at the low concentration of 0.01 mM. The presence of of KRKRWHW in the culture medium did not result in the cell death. Even at the high concentration of 5 mM, the cytotoxicity induced by the presence of KRKRWHW was not significant; the cell viability was approximately 90%. (6) The trehalose was successfully loaded to the MEFs when the cells were cultured in the medium containing 1mM KRKRWHW and 8mM trehalose for 2h. The intracellular concentration of trehalose was around 25mM, 5 times greater than the cells cultured with the medium containing trehalose only but not containing the peptide of KRKRWHW. The peptide of KRKRWHW designed in this study can accomplish the intracellular delivery of trehalose. It provides a novel approach for intracellular delivery of trehalose into mamammlian cells.
Pages80
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/8374
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
李春龙. 新型负载海藻糖穿膜肽的设计[D]. 中国科学院研究生院,2013.
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