Miles D, Athmanathan S, Thakur A, Willcox M
The Cooperative Research Centre for Eye Research and Technology, University of New South Wales, Sydney, New South Wales, Australia.
Curr Eye Res. 2003 Mar-Apr;26(3-4):165-74. doi: 10.1076/ceyr.26.3.165.14899.
Herpes simplex virus type 1 (HSV-1) infects the cornea possibly causing blindness. The specific mechanisms of herpetic keratitis are unclear. We aimed to investigate whether HSV-1 would up- or down-regulate the apoptotic pathway of human corneal epithelial (HCE) cells.
HSV-1 infection of HCE and Vero cells was demonstrated (immunofluorescence) and apoptotic gene expression was quantified (ribonuclease protection assay). Caspase 8 protein activity (colorimetric assay) was quantified and compared to caspase 8 mRNA amounts from RPA experiments. The apoptotic index of HSV-1 infected HCE and Vero cells (apoptotic index = % of apoptotic cells in infected samples/mock treated samples) was obtained and compared to gene expression.
A down-regulation in apoptotic gene expression was observed in HSV-1 infected HCE cells in contrast to Vero cells (infected and mock treated). Caspase 8 protein levels mirrored caspase 8 mRNA levels in HSV-1 infected HCE cells. The apoptotic index also supports this down-regulation. HSV-1 infected human corneal epithelial cells and Vero cells at similar rates.
HSV-1 down-regulates the apoptotic pathway of human corneal epithelial cells. This down-regulation of apoptotic gene expression seems to be cell specific. Also infectivity is excluded in playing a role in regulation of the apoptotic pathway because HSV-1 replicated at similar rates in HCE and Vero cells.
1型单纯疱疹病毒(HSV-1)可感染角膜,可能导致失明。疱疹性角膜炎的具体机制尚不清楚。我们旨在研究HSV-1是否会上调或下调人角膜上皮(HCE)细胞的凋亡途径。
通过免疫荧光法证实HSV-1对HCE和Vero细胞的感染,并通过核糖核酸酶保护试验对凋亡基因表达进行定量。对半胱天冬酶8蛋白活性进行比色测定,并与核糖核酸酶保护试验中半胱天冬酶8 mRNA的量进行比较。获得HSV-1感染的HCE和Vero细胞的凋亡指数(凋亡指数=感染样品中凋亡细胞的百分比/模拟处理样品),并与基因表达进行比较。
与Vero细胞(感染和模拟处理)相比,在HSV-1感染的HCE细胞中观察到凋亡基因表达下调。在HSV-1感染的HCE细胞中,半胱天冬酶8蛋白水平与半胱天冬酶8 mRNA水平一致。凋亡指数也支持这种下调。HSV-1以相似的速率感染人角膜上皮细胞和Vero细胞。
HSV-1下调人角膜上皮细胞的凋亡途径。这种凋亡基因表达的下调似乎具有细胞特异性。此外,由于HSV-1在HCE和Vero细胞中以相似的速率复制,因此排除了感染性在凋亡途径调节中发挥作用的可能性。