基质刚性影响声孔效应介导的单细胞基因转染

Matrix rigidity affects sonoporation mediated gene delivery on single cell

为了深入理解基质的物理特性对单细胞基因转染的影响规律,实验研究了基质刚性对单细胞质粒DNA转染效果的影响。实验采用高声压短脉冲(0.45 MPa,10μs)条件的超声对培养在不同硬度凝胶基质(软的凝胶基质:0.2 kPa,硬的凝胶基质:40 kPa)上的力学敏感细胞NIH 3T3进行质粒DNA转染实验。实验结果表明,培养在硬的凝胶基质上的细胞,质粒DNA转染效率明显高于培养在软的凝胶基质上的细胞。进一步对质粒DNA进行荧光示踪可知培养在不同刚性基质上的细胞导入质粒DNA的方式不同。当细胞被培养在硬的凝胶基质上时,通过声致穿孔产生的小孔进入细胞内的质粒DNA更多,而培养在软的凝胶基质上的细胞,更多的质粒DNA可以通过非声致穿孔作用。细胞骨架蛋白分布规律表明,硬的凝胶基质上培养的细胞内有更多的F肌动蛋白微丝,可以更好地支撑起细胞的铺展形态,相对不容易发生内吞作用。而软的凝胶基质上培养的细胞内F肌动蛋白则更多以球形状态存在,细胞形貌骗向圆形,此时更容易发生胞吞作用。

In order to acquire a deep understanding of the influence of physical properties of the extracellular matrix on single cell gene transfection,this research studies the effects of matrix rigidity on single-cell plasmid DNA transfection.NIH 3T3 cells were cultured on hydrogels with different rigidities(soft gel substrate:0.2 kPa,hard gel substrate:40 kPa),and a high-pressure short-pulse(0.45 MPa,10μs)ultrasound was used for gene transfection experiment.The experimental results show that the plasmid DNA transfection efficiency of cells cultured on a hard substrate is significantly higher than that of cells cultured on a soft gel matrix.Further fluorescence tracing of plasmid DNA shows that DNA can be intracellular uptake through separate ways for cells cultured on substrates with different rigidities.When cells are cultured on a rigid substrate,more plasmids enter the cytoplasm through cell membrane pores generated by sonoporation,while for cells cultured on a soft substrate,more plasmid DNA can be intracellular uptake through other ways.More F-actin microfilaments in cells cultured on rigid substrate supported the spreading morphology,while F-actin turned out to be clusters in cells on soft substrate which made it much easier for endocytosis.