哺乳动物呼肠孤病毒在不同细胞中的复制动力学及体外抗肿瘤活性

Replication kinetics and antitumor activity in vitro of mammalian reovirus

目的探讨哺乳动物呼肠孤病毒3型标准株(Reovirus 3)在不同细胞中的复制动力学,并对其体外抗肿瘤活性进行初步的研究。方法将低浓度的呼肠孤病毒分别接种于Vero-E6、MRC-5和MDBK 3种细胞,盲传3代,逐日观察细胞病变(CPE);采用直接免疫荧光抗体法检测盲传3代后的病毒复制情况;将黑色素瘤细胞与MDBK细胞置同一个器皿中用呼肠孤病毒进行攻毒,每日观察CPE。结果接种低浓度的呼肠孤病毒后72 h,即可在MDBK细胞中观察到明显的CPE;在MRC-5细胞中盲传3代未发现CPE;在Vero-E6细胞中盲传3代可发现轻微的CPE。呼肠孤病毒在MDBK细胞中可稳定传代;在MRC-5细胞中盲传3代未检测到病毒;在Vero-E6细胞中盲传2代即可检测到病毒,但荧光强度较弱。接种呼肠孤病毒后30 h,黑色素瘤细胞出现破裂且大面积死亡,MDBK细胞局部区域开始出现CPE,且病变区域逐渐扩大。结论 MDBK细胞是最适合培养呼肠孤病毒的细胞,Vero-E6细胞中病毒可以繁殖,但是复制动力较MDBK细胞弱,MRC-5细胞不适于培养呼肠孤病毒。呼肠孤病毒在体外具有选择性的杀死癌细胞的特性,但由于机体的免疫系统相对体外更加复杂,体内具有的抗肿瘤活性尚需在肿瘤动物模型中进行进一步的研究。

Objective To investigate the replication kinetics of standard strain of mammalian reovirus type 3 （Reovirus 3 ） in various cells and its anfitumor activity in vitro. Methods Reovirus 3 at low concentrations were inoculated to Veto- E6, MRC-5 and MDBK cells for three blind passages, observed for CPE daily and determined for replication by direct IFA. Melanoma cells and MDBK cells were placed in a vessel for virus challenge, and observed for CPE daily. Results Obvious CPE was observed in MDBK cells 72 h after inoculation with Reovirus 3 at a low concentration, while no CPE in MRC-5 cells and only slight CPE in Veto-E6 cells, after three blind passages. Reovirus 3 was subcultured stably in MDBK cells. No virus was detected in MRC-5 cells after three blind passages. However, the virus was detected in Vero- E6 cells after two blind passages, of which the fluorescence intensity was low. Melanoma cells were broken and died in a large quantity 30 h after inoculation with Reovirus 3, while local CPE in MDBK ceils, and the pathogenic area was expanded. Conclusion MDBK cells were optimal for the culture of reovirus. However, the virus might be propagated in Vero-E6 cells, of which the replication kinetics were weak than that in MDBK cells. MRC-5 cells were unsuitable for culture of reovirus. Reovirus selectively killed cancer cells in vitro, of which the in vivo antitumor activity should be further studied in animal model because of more complicated immune system.