雷帕霉素对柯萨奇病毒B3诱导的心肌成纤维细胞eIF-4E表达的影响

Rapamycin affects eIF- 4E expression in rat myocardial fibroblasts infected by Coxsackievirus B3

摘要:

目的：探讨雷帕霉素对柯萨奇病毒B3诱导的心肌成纤维细胞eIF-4E表达的影响，寻找病毒性心肌炎的药物治疗靶点。方法：选用MOI为0.5 PFU/cell的柯萨奇病毒B3感染心肌成纤维细胞，利用细胞形态及RT-PCR法检测细胞内柯萨奇病毒B3 mRNA表达，确定柯萨奇病毒B3成功感染心肌成纤维细胞。以10nM雷帕霉素干预，采用RT-PCR及Western blot检测对照组、病毒组(MOI为0.5 PFU/cell)、雷帕霉素组及病毒+雷帕霉素组eIF- 4E表达。结果：MOI为0.5 PFU/cell的柯萨奇病毒B3感染心肌成纤维细胞，透射电镜下可见细胞形态改变，胞浆内可见病毒颗粒。RT-PCR结果显示：感染后第1天、第2天、第3天及传代后第2天细胞有病毒mRNA表达。RT-PCR检测上述4组的eIF- 4E /β-actin 光密度比值分别为：0.73±0.07，0.87±0.03，0.32±0.03，0.56±0.04；Western blot灰度值分别为：0.79±0.09，1.35±0.12，0.55±0.04，0.62±0.07；RT-PCR及Western blot结果显示：病毒组eIF- 4E表达高于对照组，雷帕霉素组和病毒+雷帕霉素组低于对照组及病毒组（均P<0.05）。结论：柯萨奇病毒B3可感染心肌成纤维细胞，并使eIF- 4E表达增加，雷帕霉素可抑制其表达，提示心肌成纤维细胞可能通过mTOR/eIF- 4E信号通路参与病毒性心肌炎的发病机制，雷帕霉素对病毒性心肌炎有潜在的治疗作用。［中国当代儿科杂志，2007，9（6）：587-590］

Abstract:

OBJECTIVE: This study examined the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), on eukaryotic initiation factor (eIF- 4E) expression in rat myocardial fibroblasts infected by Coxsackievirus B3 (CVB3) in order to identify the drug target for treatment of viral myocarditis. METHODS: Primary cultured rat myocardial fibroblasts were treated with CVB3 with multiplicity of infection (MOI=0.5 PFU/cell). The experiment consisted of four groups in which the cultured rat fibroblasts cells were treated with CVB3, rapamycin (10 nM) and CVB3 + rapamycin or placebo (control). Experimental model of CVB3-infected myocardial fibroblasts was confirmed by detection of CVB3 mRNA expression with RT-PCR and observation of morphological changes of the infected cells with microscopy. eIF-4E expression was determined by both RT-PCR and Western Blot methods. RESULTS: Morphological changes were found in the fibroblasts treated with MOI 0.5 PFU/cell of CVB3 by transmission electron microscope and the viral particles were found in the cytoplasm. CVB3 mRNA was expressed in CVB3-infected fibroblasts after 1, 2, and 3 days after infection and 2 days after passage. The gray scale values of the eIF- 4E /β-actin in the control, the CVB3, the rapamycin and the CVB3+rapamycin groups were 0.73±0.07, 0.87±0.03, 0.32±0.03 and 0.56±0.04 respectively detected by RT-PCR, and were 0.79±0.09, 1.35±0.12, 0.55±0.04, and 0.62±0.07 respectively detected by Western blot. EIF- 4E expression in the CVB3 group was higher than that in the control group. Both the rapamycin and the CVB3+rapamycin groups had lower eIF- 4E expression than the control and the CVB3 groups. CONCLUSIONS: CVB3 can infect myocardial fibroblasts and up-regulate the eIF- 4E expression in rat myocardial fibroblasts. Rapamycin can inhibit eIF- 4E expression and may be a potential medicine for treatment of viral myocarditis. It was suspected that mTOR/eIF- 4E signal pathway in rat myocardial fibroblasts might play an important role in the pathogenesis of viral myocarditis.［Chin J Contemp Pediatr, 2007, 9 (6):587-590］