摘要
目的探讨siRNA抑制弓形虫硫氧还蛋白还原酶对弓形虫增殖的影响。方法将针对弓形虫硫氧还蛋白还原酶合成3组siRNA通过一定的电压和脉冲时间电转弓形虫,将弓形虫在电转30min后接种到MDA-MB-231细胞,24h后Giemsa染色,并拍照,光镜下计数细胞内平均的弓形虫速殖子数目;36h后采用荧光定量PCR检测硫氧还蛋白还原酶基因的相对表达量,试剂盒法测定酶的活性。结果 Giemsa染色后计数细胞内平均弓形虫速殖子数分别为(9±7)个、(13±10)个、(6±5)个、(37±17)个,RNA干扰后弓形虫硫氧还蛋白还原酶基因相对表达量siRNA001、002、003组分别为0.54±0.09、0.33±0.04、0.21±0.03,与阴性对照组0.93±0.08比较差异有统计学意义(t1=1.865,t2=1.898,t3=1.843,P<0.05),酶活性也显著下降。结论通过siRNA干扰弓形虫弓形虫硫氧还蛋白还原酶基因的表达,能抑制弓形虫在细胞内的增殖。
Objective To investigate the effect that inhibiting thioredoxin reductase with siRNA has on the proliferation of Toxoplasma gondii. Methods Three groups of siRNAs were synthesized with T.gondii thioredoxin reductase,and T.gondii was electrotransformed with a certain voltage and pulse time.After 30 minutes of electroporation,T.gondii was inoculated into MDA-MB-231 cells.After 24 hours,it was stained with Giemsa stain and photographed.The number of T.gondii tachyzoites in the cells was counted under a light microscope.After 36 hours,the relative expression of the thioredoxin reductase gene was detected using quantitative PCR,and the activity of the enzyme was determined using a kit. Results After Giemsa staining,the average number of T.gondii tachyzoites in the cells was 9±7,13±10,6±5,and 37±17.After RNA interference,the relative levels of expression of the thioredoxin reductase genes of T.gondii in groups treated with siRNA001,002,003 were 0.54±0.09,0.33±0.04,and 0.21±0.03 respectively.Relative levels of expression differed significantly(t1=1.865,t2=1.898,t3=1.843,P<0.05)from the level in the negative control group(0.93±0.08),and enzyme activity also decreased significantly.Enzyme activity in the three experimental groups and the negative control group was 0.000 441±0.000 098,0.0 002 205±0.000 084,0.000 147±0.000 049,and0.001 764±0.000 147 respectively. Conclusion siRNA inhibited the proliferation of T.gondii in cells by blocking the expression of the thioredoxin reductase gene of T.gondii.
作者
秦馨
李锴
张静
张爽
武卫华
陆合
QIN Xin;LI Kai;ZHANG Jing;ZHANG Shuang;WU Wei-hua;LU He(Depart-ment o f Pathogen Biology,Chongqing Medical University,Chongqing 400016,China;Molecular Medicine and Tumor Research Center,Chongqing Medical University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2018年第12期1368-1371,共4页
Journal of Pathogen Biology
基金
重庆市科委资助项目(No.cstc2015jcyjA1001)
