中药金叶败毒制剂抗人巨细胞病毒感染作用机理的实验研究

Experimental Study on the Antiviral Mechanism of Chinese Traditional Herb Jinyebaidu in HCMV Infection

目的探讨中药金叶败毒制剂拮抗HCMV感染的机理。方法采用体外实验的方法建立HCMV感染细胞膜型,给予金叶败毒制剂干预后,FQ-PCR法检测细胞内、维持液HCMV DNA拷贝数,放射免疫法测定TNF-α、IL-6蛋白含量,同时观察致细胞病变作用(CPE)的产生及传播,设立病毒对照组和更昔洛韦(GCV)药物对照组。结果分别于感染后24h、48h,实验组细胞内及维持液HCMV DNA拷贝数(5.35±0.581gGE/106HEL,2.78±0.32lgGE/106HEL)明显低于病毒组(6.12±0.65lgGE/106HEL,3.31±0.381gGE/106 HEL,P<0.05),持续至感染晚期,与药物对照组比较,感染晚期实验组细胞内病毒负荷量(7.97±0.81lgGE/106HEL)高于药物对照组(7.05±0.71lgGE/106HEL,P<0.05),但两者维持液内病毒量无明显差异(P>0.05)。分别于感染后12h、24h,实验组维持液TNF-α、IL-6蛋白含量(0.63±0.06pg/ml,38.26±4.19pg/ml)明显高于病毒组(0.55±0.04pg/ml,31.12±4.53pg/ml,P<0.05)和药物对照组(0.56±0.05pg/ml,32.40±4.97pg/ml,P<0.05),在感染后24～36h达高峰(P<0.01)。实验组和药物对照组CPE出现(感染后96h)均晚于病毒组(感染后48h),且发展缓慢,感染后168h仅为++,而病毒组则达到++++。结论金叶败毒制剂可有效抑制感染细胞内HCMV DNA复制及病毒自受染细胞内释放。

Objective To study the antiviral mechanism of Chinese traditional herb Jinyebaidu in HCMV infection. Methods In vitro HCMV infected cell model was established by coincubating the HEL cells and HCMV AD169 with liter of 100TCID50.The culture was then without intervention (group A)and with intervention of Jinyebaidu (group B)or GCV(group C). Intracellular and supernatant HCMV genome copies were evaluated by FQ-PCR.TNF-α and IL-6 in supernatants were detected by radioimmunoassay. Viral infection was also detected by CPE assay. Results 24h after infection, intracellular HCMV DNA replication was inhibited in group B(5.35 ± 0.581gGE/106HEL) compared with group A(6.12 ± 0.65lgGE/106HEL, P <0.05),but not so significantly as that of group C(168h postinfection; 7.05 ± 0.711gGE/106HEL,groupB:7.97±0.811gGE/106HEL, P <0.05).And virion release from infected cells was blocked 48h after infection in group B(2.78 ± 0.321gGE/106HEL, group A:3.31 ±0.381gGE/ 106/HEL, P <0.05).The viral liters of group Band group Chad no significant difference( P > 0.05) .The levels of TNF-α and IL-6 in the supernatants of group B were also elevated to 0.63 ± 0.06pg/ml, and 38.26 ± 4.19pg/ml, beginning at 12h and 24h after infection respectively. While no significant difference was showed between group A and C(group A: 0.55 ± 0.04pg/ml, 31.12 ± 4.53pg/ml,group C:0.56± 0.05pg/ml,32.40 ± 4.97pg/ml, P < 0.05) .The onset of CPE was delayed in group B and C, and showed a slower progression(96h postfecton, 168h postinfection: + + )than group A(48h postinfection, 168h postinfection: + + + + ). Conclusion Jinyebaidu shows an inhibiting effect on intracellular HCMV DNA replication and virion release from infected cells. It could also enhance the expression of TNF-αand IL-6 to regulate the function of the immune system. By these means, Chinese traditional herb Jinyebaidu has an antiviral efficacy in HCMV infection.