人源性及牛源性乳铁蛋白抑制丙型肝炎病毒复制作用的对比研究

A Comparative Study of the Depressant Effects of Human and Bovine Lactoferrins on the Replication of Hepatitis C Virus in Vitro

目的 :探讨人乳铁蛋白 (HLF)及牛乳铁蛋白 (BLF)在体外HepG2 细胞中对丙型肝炎病毒 (HCV)感染增殖的抑制作用 ,为乳铁蛋白用于临床治疗HCV感染提供理论和实验依据。方法 :以HepG2 细胞作为HCV复制体系 ,以RT nPCR方法 ,测定培养细胞提取物中病毒正、负链 ,并用活性细胞定量鉴定法 (MTT)对两种乳铁蛋白的细胞毒性作用及药物剂量进行研究。结果 :细胞毒实验显示 ,实验中两种乳铁蛋白的浓度小于 1.5mg·mL 1时对HepG2 细胞生存率无显著影响。HCV阳性血清接种HepG2 细胞后 ,HCV正、负链可于第 8,10 ,12天被检测到。当两种乳铁蛋白 (浓度分别为 1.5,1.0 ,0 .5,0 .1mg·mL 1)先与HCV阳性血清作用后再接种细胞时 ,不同时期提取物中均未测定到HCV正、负链。而当乳铁蛋白 (浓度同上 )先与细胞作用后洗去再与HCV阳性血清作用时 ,不同时期细胞提取物中可测定到HCV正、负链。结论 :乳铁蛋白能在非细胞毒性剂量时能防止HCV感染HepG2 细胞 ;乳铁蛋白可能主要是通过与病毒颗粒本身相互作用发挥上述功能 ;两种来源的乳铁蛋白在体外抑制HCV复制的作用差异无显著性。

Objective:To probe into the depressant effec ts of human lactoferrin(HLF) and bovine lactoferrin(BLF) on the replication of hep atitis C virus (HCV) in HepG 2 cell culture, so as to provide theoretical as we ll as experimental basis for the clinical management of HCV infection. Methods:HepG 2 cell culture was used as the host of HCV tranfection and replication HCV positive chain RNA and negative chain RNA were assayed with RT-nested PCR. The cytotoxic effect of the lactoferrins on HepG 2 cells was examined with the MTT test. Results:Both human and bovine lactoferrins in high conce ntrations were shown to exert cytotoxic effects on HepG 2 cells However when th e concentration of the lactoferrins was 1.5 mg·mL -1 or lower, no cytotox icity was present. Both HCV positive chain RNA and negative chain RNA could be d etected from the cell extract within several days after the HepG 2 cells had be en inoculated with HCV RNA positive serum. If HLF or BLF (at concentrations of 1 .5 mg·mL -1 , 1.0 mg·mL -1 and 0.5 mg·mL -1 ) had been co-in cubated with HCV positive serum for a period of time before addition of HepG 2 cells or co-incubated with HepG 2 cells followed by the admixture of HCV posit ive serum, no HCV positive chain RNA nor HCV negative chain RNA could be detecte d in the cell extract with RT-nested PCR. If, however the LF (at the same conce ntrations as mentioned above) co-incubated with the HepG 2 cells had been wash ed away before the addition of HCV positive serum both HCV positive chain RNA an d negative chain RNA were demonstrated in the cell extract with RT-nested PCR. Taken togethers the findings of these 3 experiments suggest that the depressant effects of the LF in non-cytotoxic concentrations on the replication of HCV in HepG 2 cells inoculated with HCV positive serum may be attributed to binding o f the LF to corresponding sites on the virus. It seems unlikly that these effec ts were caused by interaction between the LF and the HepG 2 cells. The differe nce between the effects of HLF and BLF was in-significant. Conclusion:HLF and BLF in non-cytotoxic concentrations were shown to exert a depressant effect on the replication of HCV in HepG 2 cel l culture inoculated with HCV positive serum, possibly by binding of the LF to corresponding sites on the virus.