摘要
目的研究鸦胆子油乳(brucea javanica oil emulsion,BJOE)在体外对人乳头瘤病毒(human papillomavirus,HPV)16亚型感染细胞的抑制作用及其可能作用机制。方法选择人宫颈鳞状上皮永生化细胞系(Ect1/E6E7)与人宫颈癌CaSki细胞系作为HPV16亚型感染的宫颈癌前病变与宫颈癌体外实验模型。采用MTT法检测5、10、20及40μg/mL BJOE体外分别作用24、48及72 h对HPV16感染细胞增殖活性的影响;Hoechst 33258细胞核染色荧光显微镜下观察细胞凋亡形态学变化;Annexin V-FITC/PI双染色法检测细胞凋亡率;半定量RT-PCR法检测HPV16 E6、E7基因mRNA的表达;免疫细胞化学染色(Elivison二步法)检测HPV16 E6、E7、p53及Rb基因蛋白的表达。结果 (1)5、10、20及40μg/mL BJOE体外分别作用24、48及72 h对HPV16感染细胞的增殖具有明显抑制作用,且表现为浓度与时间的依赖性(P<0.05)。(2)HPV16阳性细胞在BJOE作用后发生了细胞凋亡形态学变化,5、10及20μg/mL BJOE体外作用48 h后细胞凋亡率逐渐升高(P<0.05)。(3)5、10及20μg/mL BJOE体外作用HPV16阳性细胞48 h后E6与E7基因mRNA的相对表达量逐渐降低(P<0.05)。(4)5、10及20μg/mL BJOE作用后,HPV16 E6、E7及突变型P53蛋白的表达逐渐减弱(P<0.05),而Rb蛋白的表达逐渐增强(P<0.05)。结论 BJOE在体外对HPV16亚型感染细胞具有明显的抑制作用,其作用机制可能与药物下调病毒癌基因E6、E7表达相关。
Objective To explore the in vitro inhibitive effect and underlying mechanisms of Bru- cea Javanica oil emulsion （B JOE） on human papilloma virus （HPV） type 16 infected cells. Methods The HPV16 E6/E7 immortalized human ectocervical Ectl/E6E7 cell line and the CaSki cell line were selected as the in vitro models of premalignant cervical lesion and cervical cancer respectively. After treated with B JOE at different concentrations （5, 10, 20, and 40 μg/mL） at the operation time points （24,48, and 72 h）, the effects of B JOE on proliferative activities were measured by MI-I- assay. The morphologic changes of cell apoptosis stained with Hochest 33258 were observed by fluorescence microscope. The effect on the cell apoptosis rate was analyzed by Annexin V-FITC/PI double-labeled flow cytometry. The mRNA expressions of HPV16 E6 and E7 were determined by semi-quantitative RT-PCR. The protein expressions of HPV16 E6, E7 oncogene, and specifically interacted p53, Rb antioncogene were stained by immunocytochemical staining （Elivison two-step procedure）. Results （1） The proliferative activities of the Ectl/E6E7 cell and the CaSki cell treated with B JOE at different concentrations （5, 10, 20, and 40 μg/mL） at the operation time points （24,48, and 72 h） were obviously inhibited, showing dose-and time-dependent manners （P 〈0.05）. （2） Typical changes of apoptosis were observed in both HPV 16 positive cell lines after treated with B JOE. The cell apoptosis rates increased markedly after being cultured with B JOE at different concentrations （5, 10,and 20μg/mL） for 48 h （P 〈0.05）. （3） After treated with B JOE at different concentrations （5, 10, and 20 μg/mL） for 48 h, the HPV16 E6 and E7 mRNA relative expressions in both HPV 16 positive cell lines de- creased significantly （P 〈0.05）. （4） After treated with B JOE at different concentrations （5, 10, and 20 μg/ mL）, the expressions of HPV16 E6, E7, and mutant p53 protein decreased gradually （P 〈0.05）, while the Rb protein expression increased gradually （P 〈0.05）. Conclusions B JOE showed obvious in vitro inhibito- ry effects on HPV type 16 infected cells. Its underlying mechanisms might be possibly associated with down-regulating expressions of HPV16 E6 and E7 oncogenes.
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
2013年第11期1545-1551,共7页
Chinese Journal of Integrated Traditional and Western Medicine
基金
温州市科技计划资助项目(No.Y20090261)
关键词
鸦胆子油乳
宫颈
人乳头状瘤病毒
凋亡
Brucea Javanica Oil Emulsion
cervix
human papilloma virus
apoptosis
作者简介
通讯作者：胡燕，Tel：13806696807，E-mail=627830566@qq．com
