日本血吸虫机械脱尾童虫体外转染siRNA的效率研究  

Efficiency of siRNA transfection into Schistosoma japonicum schistosomula in vitro with different methods

作　　者：赵静[1] 林超[1] 潘卫庆[1] 

机构地区：[1]同济大学生命科学与技术学院,上海200092

出　　处：《同济大学学报（医学版）》2015年第3期1-5,共5页Journal of Tongji University(Medical Science)

基　　金：国家"973"重点基础研究发展计划(2007CB513100)

摘　　要：目的比较体表渗透法、电穿孔法、脂质体转染试剂LipofectamineTM2 000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 （ LipofectamineTM 2 000 ） 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^TM2 000 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.

关 键 词：日本血吸虫 机械脱尾童虫 SIRNA 转染效率 

分 类 号：R73[医药卫生—肿瘤]