鸦胆子油乳对HPV16亚型感染细胞的作用及机制研究

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40 μg/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40 μg/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20 μg/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20 μg/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20 μg/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。

英文摘要:

ObjectiveTo explore the in vitro inhibitive effect and underlying mechanisms of Brucea Javanica oil emulsion （BJOE） on human papilloma virus （HPV） type 16 infected cells. MethodsThe HPV16 E6/E7 immortalized human ectocervical Ect1/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 BJOE at different concentrations （5， 10， 20， and 40 μg/mL） at the operation time points （24， 48， and 72 h）， the effects of BJOE on proliferative activities were measured by MTT 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 Ect1/E6E7 cell and the CaSki cell treated with BJOE 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 BJOE. The cell apoptosis rates increased markedly after being cultured with BJOE at different concentrations （5， 10， and 20 μg/mL） for 48 h （P<0.05）. （3） After treated with BJOE 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 decreased significantly （P<0.05）. （4） After treated with BJOE 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）. ConclusionsBJOE showed obvious in vitro inhibitory effects on HPV type 16 infected cells. Its underlying mechanisms might be possibly associated with down regulating expressions of HPV16 E6 and E7 oncogenes.

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