新型纳米自组装短肽KVDV-16作为生物支架材料的可行性

Feasibility of new self-assembling peptide KVDV16 as a biological scaffold material

背景:设计合成和构建纳米生物材料如纳米自组装短肽是以一种"从下而上"的方式完成,其在生物医学工程方面潜在的应用价值引起了众多研究团队越来越广泛的兴趣。目的:初步探讨纳米自组装短肽KVDV16水凝胶在细胞三维培养方面的可行性。设计、时间和地点:体外观察实验,于2007-11/2008-11在四川大学纳米生物医学技术与膜生物学研究所纳米生物实验室进行。材料:短肽KVDV16(纯度为95%,为了保护肽的两端,对其N端和C端分别进行乙酰化和酰胺化);人肝细胞系L-02和肝癌细胞系SMMC7721。方法:圆二色谱仪和傅里叶红外光谱检测短肽KVDV16水凝胶在不同理化条件下的二级结构;原子力显微镜检测其水凝胶形成的纳米级结构特征;扫描电子显微镜观察其形成的纳米三维支架。主要观察指标:β折叠二级结构,纳米纤维结构特征,细胞在肽支架材料中的生长、增殖情况。结果:短肽KVDV16形成的β折叠二级结构在高温下很稳定。在不同的pH(3,7,10)甚至变性剂1%十二烷基磺酸钠的作用下,β折叠二级结构也只发生轻微的变化。原子力显微镜证实其由纳米纤维结构组成。L-02和SMCC7721细胞进入了KVDV16支架材料,镶嵌在纳米纤维网中,并很好地在此三维环境中生长。结论:实验设计的短肽KVDV16是对自组装系统的一个补充,此短肽表现出来的性质使其能发展成为组织工程中细胞培养的三维支架材料。

BACKGROUND： Designing, synthesizing and fabricating biological nano-materials, such as nanofiber scaffolds, based on a ＂bottom-up＂ self-assembling peptide system, have been attracting great interest of numerous groups for many potential important applications in both academia and biomedical engineering OBJECTIVE： To investigate the feasibility of a self-assembling peptide KVDV16 hydrogel scaffold for three-dimensional cell culture. DESIG,TIME AND SETTING：An in vitro observational study was performed in Institute for Nanobiomedical Technology and Membrane Biology, Sichuan University from November 2007 to November 2008. MATERIALS： Peptide KVDV16 （purity： 95%; acetylation and amidation were performed at N and C terminals, respectively）, cell lines L02, and SMCC7721 were used in this study. METHODS： Circular dichroism spectra and Fourier transform infrared spectroscopy were used to detect the secondary structure of peptide KVDV16 diluted in water. Atomic force microscope was used to detect the nanostructure characteristics formed by KVDV16. The three-dimensional environment for cell culture formed by KVDV16 was detected by scanning electron microscopy. MAIN OUTCOME MEASURES： β -sheet secondary structure, performance of nano-fiber structure, cell growth and proliferation on peptide scaffold. RESULTS The .β -sheet secondary structure was very stable at high temperature. Various pH changes at 3, 7 and 10, there was a few effects on the stability of the 13 -sheet secondary structure, even in the presence of 1% sodium dodecylsulphate. Furthermore, atomic force microscope indicated that the .β -sheet secondary structure consisted of nano-fibers. L-02 and SMCC7721 entered the KVDV16 scaffold and were located in nano-fiber net and grew well in three-dimensional environment. CONCLUSION： The new peptide KVDV16 can be a complementarity to the self-assembling system and also its properties studied here make it to develop a biological matrix scaffold for cell attachment in tissue engineering.