鸦胆子素D对HPV16感染细胞株的作用及其可能机制

The effect of Bruceine D on high-risk human papillomavirus 16 infected cell and its possible mechanism

目的:研究鸦胆子素D对高危型人类乳头瘤病毒(HPV)16感染细胞的增殖抑制作用、促凋亡作用及其可能机制。方法:以人宫颈鳞状上皮永生化细胞株(Ect1/E6E7)作为HPV16型感染的宫颈癌前病变的体外实验模型,用1、5、10、15、30μmol/L的鸦胆子素D分别体外作用于细胞24、48、72 h,用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法测定细胞增殖活性改变;以1、5、10μmol/L的鸦胆子素D分别体外作用于细胞24、48、72 h后,Cell cycle staining solution检测细胞生长周期变化,Hoechst33258细胞核荧光染色法观察细胞凋亡情况,Annexin V-FITC/PI双染色法检测细胞凋亡率。宫颈癌细胞株Caski含有完整的HPV16型E6、E7基因序列,以此作为阳性对照,通过实时荧光定量PCR技术检测两种细胞内HPV16 E6、E7基因m RNA的表达。结果:鸦胆子素D对Ect1/E6E7细胞具有明显的抑制体外增殖作用,MTT结果显示抑制作用呈时间和浓度依赖性(P<0.05)。经1、5、10μmol/L鸦胆子素D体外作用48 h后,细胞生长周期停滞于G1期。流式细胞仪检测显示,各实验组细胞经鸦胆子素D作用48 h后,细胞凋亡率分别为(6.25±0.76)%、(20.21±1.32)%和(8.61±1.59)%,与对照组比较差异有统计学意义(P<0.05)。Hochest33258细胞核染色法显示细胞出现凋亡形态学改变。Real-time PCR技术检测结果显示Ect1/E6E7中HPV16 E6、E7m RNA表达水平无明显下降,与对照组比较差异无统计学意义(P>0.05)。而Caski中HPV16 E6、E7 m RNA表达水平下降,与对照组比较差异有统计学意义(P<0.05)。结论:鸦胆子素D能有效地抑制Ect1/E6E7细胞增殖,其机制可能是通过抑制HPV16 E6、E7 m RNA表达从而促进细胞凋亡。

infected cell in vitro. Methods：The Ect1/E6E7 was cultivated with BD （1, 5, 10, 15 and 30μmol/L） for 24, 48 and 72 h, the anti-proliferation effect was measured with MTT colorimetric assay;The Ect1/E6E7 was cultivated with BD （1, 5 and 10μmol/L） for 24, 48 and 72 h, the cell growth cycle change was measured with cell cycle staining solution, the morphologic changes of cell apoptosis stained with Hoechst 33258 in the Ect1/E6E7 cell line were observed under lfuorescence microscope treated with BD at concentration of 5μmol/L for 48 h. Flow cytometry of Annexin V-FITC/PI assay was used to evaluate the apoptosis rate of Ect1/E6E7 cell line treated with BD at different concentrations （1, 5 and 10μmol/L） in vitro. Cervical cancer cells Caski which contain complete HPV 16 E6, E7 gene, as a positive control, by real-time quantitative PCR to detect the mRNA expression of HPV E6, E7 of those two cells treated with BD. Results：Immortalized human ectocervical Ect1/E6E7 cell line treated with BD showed obviously morphological changes, and with the prolongation of drug action and the increase of drug concentration, the morphological changes became more apparent. BD can inhibit the proliferation of Ect1/E6E7 cell in dose-dependent and time-dependent manner （P〈0.05）. Ect1/E6E7 cell growth cycle was stag-nated in the G1 phase. Hochest lfuorescence staining presented the Ect1/E6E7 cells nucleus appeared apoptotic morphological change, and apoptotic bodies can be observed. Flow cytometry showed the apoptosis rate were＆amp;nbsp;（6.25±0.76）%, （20.21±1.32）%, （8.61±1.59）%, respectively, after 48 hours cultured with 1, 5, 10μmol/L of BD （P〈0.05）. Real-time PCR results showed that after cultivating with 1, 5, 10μmol/L BD for 48 h, the expression of HPV16 E6, E7 mRNA of Ect1/E6E7 cell were decreased, but compared with the control group, the difference was not statistically signiifcant （P〉0.05）. The expression of HPV16 E6, E7 mRNA of Caski cell were decreased, the difference was statistically signiifcant （P〈0.05） compared with the control group. Conclusion：BD can inhibit the proliferation of Ect1/E6E7 cells signiifcantly, the possible mechanism may be associated with the expression decline of HPV16 E6, E7 mRNA, which promote cell apoptosis.