CAJ | 학술논문

目的：探讨转录因子p53在登革病毒感染中的作用。方法用逆转录病毒载体介导的RNA干扰技术构建p53低表达的人肝癌细胞株HepG2，并采用Western blot 进行鉴定。设定野生组和干扰组，分别用2型登革病毒感染。采用Vero细胞噬斑法检测病毒滴度；间接免疫荧光检测病毒增殖；流式细胞术检测病毒感染后细胞凋亡；酶联免疫吸附试验检测IFN-β分泌。结果与野生组比较，干扰组p53表达明显下调，表明构建p53低表达的HepG2细胞株成功。登革病毒感染24 h后，干扰组病毒滴度比野生组高100倍；免疫荧光显示干扰组发绿色荧光的细胞数明显多于野生组，说明p53抑制了登革病毒的感染。但是，登革病毒感染24 h后两组凋亡细胞的数目并没有明显差异，而野生组IFN-β分泌量增加6倍。结论转录因子p53抑制登革病毒感染可能不是通过促进细胞凋亡，而是通过激活Ⅰ型干扰素通路来发挥作用。
목적：탐토전록인자p53재등혁병독감염중적작용。방법용역전록병독재체개도적RNA간우기술구건p53저표체적인간암세포주HepG2，병채용Western blot 진행감정。설정야생조화간우조，분별용2형등혁병독감염。채용Vero세포서반법검측병독적도；간접면역형광검측병독증식；류식세포술검측병독감염후세포조망；매련면역흡부시험검측IFN-β분비。결과여야생조비교，간우조p53표체명현하조，표명구건p53저표체적HepG2세포주성공。등혁병독감염24 h후，간우조병독적도비야생조고100배；면역형광현시간우조발록색형광적세포수명현다우야생조，설명p53억제료등혁병독적감염。단시，등혁병독감염24 h후량조조망세포적수목병몰유명현차이，이야생조IFN-β분비량증가6배。결론전록인자p53억제등혁병독감염가능불시통과촉진세포조망，이시통과격활Ⅰ형간우소통로래발휘작용。
Objective To investigate the role of a transcription factor p 53 in dengue virus infec-tion.Methods A plasmid expressing siRNA specific for p 53 gene was constructed and then used to prepare HepG2 cell line with a suppressed expression of p 53 protein.The expression of p53 protein was detected by Western blot assay .A wild type control group and a siRNA group were set up by infecting wildtype HepG 2 cells and p53 low expressing HepG2 cells with type 2 dengue viruses,respectively.The virus titers in two dif-ferent cells were determined by plaque forming assay using Vero cells .Indirect immunofluorescence assay was performed to detect virus multiplication .The apoptosis of virus infected cells were analyzed by flow cytome-try.ELISA was performed to analyze the levels of IFN-βsecreted by infected cells from two groups .Results Compared with wildtype control group ,the cells in siRNA group showed a suppressed expression of p 53 pro-tein,suggesting that the HepG2 cell line with low p53 protein expression was successfully established .The vi-rus titer in supernatants of the cells from siRNA group was about 100-fold higher than that of wildtype control group at 24 hours after viral infection .Fluorescence activated cell sorting analysis showed that the numbers of green fluorescence labeled cells were remarkably increased in siRNA group .We speculated that p53 protein might play a role in the inhibition of dengue virus infection as indicated by the observed results .The numbers of apoptotic cells showed no significant difference between two groups .However,the level of IFN-βsecreted by wildtype HepG2 cells was six times higher than that of the cells in siRNA group .Conclusion p53 pro-tein might inhibit dengue virus infection through the activation of type Ⅰ interferon signaling pathway rather than enhance cell apoptosis .