日本血吸虫机械脱尾童虫体外转染siRNA的效率研究

目的 比较体表渗透法、电穿孔法、脂质体转染试剂Lipofectamine TM2000以及纳米材料聚乙烯亚胺(PEI)4种不同的方法用于日本血吸虫机械脱尾童虫体外转染siRNA的转染效率,以期筛选理想的转染方法。方法 选用携带有红色荧光标记的化学合成siRNA,并且据根不同转染方法说明步骤优化体外转染条件,分别转染日本血吸虫机械脱尾童虫。在一定的时间内利用荧光显微镜观察虫体转染情况并计阳性虫数,并应用反转录聚合酶链反应(RT-PCR)技术检测转入靶基因的mRNA表达情况。结果 经过体外转染条件的优化,电穿孔和纳米材料介导的siRNA转染效率达到了90％以上。应用RT-PCR验证,4种方法中,电穿孔转染法和纳米材料转染的siRNA对靶基因有显著的抑制效应(P＜0.05),电穿孔法转染抑制率达(45±9.63)％,纳米材料转染抑制率达(37±6.17)％。结论 除了常规的电穿孔法,纳米材料作为新型的转染载体,能够有效地传递siRNA进入血吸虫童虫体内,干扰目的基因的表达,这将为日本血吸虫功能基因学的研究提供高效的转染工具。
Objective To compare the efficiency of siRNA transfection to schistosoma japonicum schistosomula in vitro by different methods. Four methods are soaking, electroporation, liposome transfection reagents(LipofectamineTM2000) and nano-materials polyethyleneimine(PEI). Methods“Mechanical schistosomula”were prepared by mechanical transformation of Schistosoma japonicum cercaria. Chemical-synthesized siRNA with red fluorescent tags was transfected to mechanical-transformed shistosomula by 4 different methods:soaking, electroporation, LipofctamineTM2000 and nano-polyethyleneimine(nano-PEI), respectively. The transfection efficiency was observed by fluorescence microscope, the mRNA expression of target genes was detected by reverse transcription polymerase chain reaction(RT-PCR). Results The transfection efficiency was more than 90% mediated by electroporation and nano-PEI under optimal transfection conditions in vitro. Target genes had a significant inhibitory effect (P＜0.05) with a inhibited rate of(45±9.63)% for electroporation and(37±6.17)% for nano-PEI. Conclusion In addition to the commonly used electroporation, nano-PEI as a new type of transfection vector can effectively transfect the target gene in mechanical-transformed shistosomula of Schistosoma japonicum