疏水蛋白HGFI用于载基因组织工程支架的研究  

The Study of Fungal Hydrophobin HGFI on Gene Delivery Tissue Engineering Scarfford

作　　者：张沛欣 马绍扬 乔明强[1] 张钧[1] Zhang Peixin;Ma Shaoyang;Qiao Mingqiang;Zhang Jun(College of Life Sciences,Nankai University,Tianjin 300071,China)

机构地区：[1]南开大学生命科学学院,天津300071

出　　处：《南开大学学报（自然科学版）》2018年第5期1-7,共7页Acta Scientiarum Naturalium Universitatis Nankaiensis

基　　金：国家自然科学基金(81471799)

摘　　要：以具有两亲性的疏水蛋白HGFI为基础,通过正负电荷的相互作用将质粒与PEI的复合体固定在了修饰有疏水蛋白HGFI的PCL支架材料上.对不同时间段释放的质粒进行了定量测量并绘制曲线,结果表明载基因支架上的基因有四分之三左右在固定的12 h内释放.用载基因支架进行体外293T的转染实验,载基因支架的转染效率为21.76%.该转染效率与直接用转基因试剂对贴壁细胞转染的效率低很多,但对于组织工程支架来说,已具有一定的基因递送功能.大鼠皮下埋植实验表明,在体内埋植3 d后,载基因支架有着良好的体内转染效果,对体内的转基因阳性细胞进行初步检测发现,支架埋植初期有大部分被转染细胞为巨噬细胞,而对巨噬细胞进行转染是否对组织修复有益还需进一步的研究.大鼠皮下埋植2 w后,对埋植的材料进行血管化的检测,组织切片进行vWF免疫荧光染色和成像,成像结果说明载基因支架显著地促进了支架材料在体内的血管新生,推测支架上固定的质粒在体内转染后表达出了目的蛋白e NOS,从而促进了支架材料周围组织的血管化.本实验中,载基因支架的转染效率还需进一步提高,体内转基因阳性细胞的种类还需进行进一步鉴定,功能蛋白eNOS的表达和检测还需要进一步的研究.Fungal hydrophobin HGFI is a kind of small molecular weight amphiphilic proteins which have both hydrophilic and hydrophobic structure.On the hydrophobic surface of PCL materials, HGFI can form a layer of protein by self-assembling and change the surface of PCL to hydrophiic. hydrophobi HGFI is studied as gene carrier in the tissue engineer scaffold. Under physiological conditions HGFI is negatively charged. When the N/P ration of PEI-DNA complex is over 2, the complex is electropositive. DNA can be immobilized on the HGFI modified PCL materials by charge interaction. The plasmid in this study expresses green fluorescent protein（GFP） and endothelial nitric oxide synthetase（eNOS）. The expression of GFP reports the complete of the gene delivery which can assayed by flow cytometry and fluorescence imaging, Labled the plasmid with fluorescence confirms whether the plasmid was immobilized on the scaffold.The rat subcutaneous implant model is applied to evaluate the effect of gene delivery. The results of the experiments show that plasmid can be immobilized on the PCL scaffold through HGFI, and deliver the gene to cells in vitro and in vivo and play the function.

关 键 词：疏水蛋白 转基因 组织工程 电纺丝 ENOS 

分 类 号：Q819[生物学—生物工程]