siRNA干扰嘌呤补救合成途径的关键酶对弓形虫增殖的抑制作用  

siRNAs specific to key enzymes in the purine salvage pathway retard growth of Toxoplasma gondii

作　　者：余莉[1] 李霞[2] 郜玉峰[3] 罗庆礼[1] 乔增培[1] 沈继龙[1] 

机构地区：[1]安徽医科大学病原生物学教研室,人兽共患病安徽省重点实验室,教育部省部共建重点实验室,合肥230032 [2]安徽中医学院第二附属医院,合肥230061 [3]安徽医科大学第一附属医院感染病科,合肥230032

出　　处：《中国人兽共患病学报》2009年第1期9-12,16,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金项目(30800965);安徽省教育厅高校自然科学基金(KJ2008B291)

摘　　要：目的探讨siRNA同时干扰嘌呤补救合成途径中两个关键酶对弓形虫增殖速率的影响。方法将针对腺苷激酶(AK)和次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖转移酶(HXGPRT)的siRNA同时电转染弓形虫,然后将弓形虫接种人包皮成纤维细胞(HFF),24h和40h后Giemsa染色,光镜下计数假包囊内弓形虫速殖子数目,计算弓形虫平均倍增次数。同时将弓形虫进行模拟电转,接种HFF细胞后,不加磺胺嘧啶进行培养作为阴性对照,加入10μg/mL磺胺嘧啶培养作为阳性对照。结果在接种HFF细胞后24h,4μmol/L的AK siRNA786和HXGPRT siRNA415联合电转弓形虫、阳性对照组及阴性对照组弓形虫的平均倍增次数分别为:1.64±0.95、1.04±0.88及2.32±0.90,前两组与后者相比倍增次数明显减少(P<0.05)。在接种后40h,siRNA联合电转弓形虫与阳性对照组弓形虫的平均倍增次数分别为3.00±1.04,2.56±1.04,与阴性对照组相比(3.60±0.80)差异有显著性(P<0.05)。结论嘌呤补救合成途径中的两个关键酶-AK和HXGPRT被其siRNA同时抑制后,弓形虫在细胞内的增殖速率显著减缓。Adenosine kinase （AK） and hypoxanthine-xanthine-guanine phosphoribosyltransferase （HXGPRT） are the two key enzymes involved in the purine salvage pathway in T. gondii. We targeted the genes encoding AK and HXGPRT in T. gondii by exposing the parasites to siRNAs encoding part of the coding regions. When 4μmol/L AK siRNA786 was electroporated into the parasites, the treated parasites exhibited significantly lower AK activity compared with the parasites of the mock was simultaneously targeted eleetroporation after 24hrs treatment, and the relative values of ^3 H-adenosine incorporations in parasites decreased to 0.39 ± 0.11. When 4 μmol/L HXGPRT siRNA415 was electroporated into the parasites 24hrs post-in- fection, the relative value of HXGPRT activity decreased to 0.51±0.03. To observe the effect of siRNA treatment on T. gondii proliferation, the genes encoding AK and HXGPRT in T. gondii by using siRNA. After electroporation with 4μmol/L of AK siRNA786 and HXGPRT siRNA415, the parasites were inoculated onto the HFF monolayer. The average doubling for siRNA-treated, SDZ-treated and mock-treated parasites was 1.64±0.95, 1.04±0.88 and 2.32± 0.90, respectively, 24 hrs post-inoculation. In comparison with mock-treated parasites, the rate of replication for siRNA-treated and SDZ-treated was significantly slowed down（P〈0.05）. At 40hrs after inoculation, the average doubling for siRNA-treated and SDZ-treated was 3.00± 1.04 and 2.56± 1.04,respectively, and significantly lower than the values （3.60 ± 0.80） obtained from mock-treated parasites at the same time points （P〈0.05）. The data show that transfection of siRNAs into cells can efficiently regulate gene expression in T. gondii. When the genes encoding AK and HXGPRT were inhibited simultaneously using siRNAs, the rate of replication of T. gondii was significantly slowed down.

关 键 词：弓形虫 嘌呤代谢 腺苷激酶 次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖转移酶 RNA干扰 SIRNA 

分 类 号：R531.8[医药卫生—内科学]