cDNA microarray analysis of toxoplasmic retinochoroiditis

利用自制的基因芯片系统，利用正常小鼠的mRNA构建了约15,000个克隆，对弓形虫感染诱导的基因进行了分析，获得了5个基因群：β2-微球蛋白、DMR-N9、第14点、睾酮和一个未知基因(暂定为T.g.Res.X)。通过比较WT耐药株(BALB/c)和WT敏感株(C57BL/6)以及WT和干扰素-γ基因敲除(GKO)BALB/c小鼠之间的差异，证明T.g.Res.X在弓形虫感染抗性株中表达。分析包括眼科检查、荧光血管造影术、定量竞争聚合酶链式反应、干扰素-γ逆转录聚合酶链式反应、阶段转化标志物和cDNA微军团分析。弓形虫载量以视网膜后部最高，远高于WT BALB/c小鼠。在GKO小鼠中，播散性感染很明显，脉络膜中的弓形虫载量最高，等等。WT C57BL/6小鼠感染后干扰素-γ基因表达水平高于WT BALB/c小鼠。GKO小鼠体内存在速殖子，而WT C57BL/6小鼠体内存在缓殖子，GKO小鼠视网膜毛细血管内染料渗出。与耐药品系不同，敏感WT品系小鼠视网膜弓形虫载量持续增加。干扰素-γ对弓形虫在眼内的载量和相互转换有调节作用。用GKO小鼠建立弓形虫血管炎模型，建立了QC-PCR和FA3种检测系统，并利用基因芯片系统对弓形虫视网膜脉络膜炎相关基因进行了鉴定

By using our own made cDNA microarray system composed with around 15,000 clones established by using mRNA from normal mice, Toxoplasma gondii-infection-induced genes were analyzed.Five gene groups, beta2-microglobulin, DMR-N9, spot 14,teststeron, and an unknown (tentatively named as T.g.Res.X) were picked up. T.g.Res.X was shown to express in a resistant strain to T.gondii infection when the mRNA expression was examined by comparing between a WT resistant strain (BALB/c) and a WT susceptible strain (C57BL/6) and also comparing between WT and IFN-γ knockout (GKO) BALB/c mice.WT C57BL/6, WT BALB/c, and GKO mice were infected with cysts of T.gondii perorally. Analysis included ophthalmic examinations, fluorescein angiography (FA), a quantitative competitive polymerase chain reaction (QC-PCR) assay, reverse transcription (RT)-PCR for IFN-γ and stage conversion markers and cDNA microarmy assayIn WT C57BL/6 mice, T.gondii was detected in the order Of the brain, retina, choroid, sclera end optic nerve (ON). The highest T.gondii load was observed in the posterior retina, and was much greater than that in WT BALB/c mice. In GKO mice, disseminated infection was evident and the T.gondii load was highest in the choroid, and ON. IFN-γ mRNA expression in WT C57BL/6 mice was higher than that in WT BALB/c mice after infection. Tachyzoites existed in GKO mice, while bradyzoites existed in WT C57BL/6 mice FAG showed dye leakage from the retinal capillaries of GKO mice.The T.gondii load in the retina in the susceptible WT strain continued to increase, unlike in the resistant WT strain. IFN-γ was shown to regulate the T gondii load and interconversion in the eye. A toxoplasmic vasculitis model was established with GKO mice and assay systems with QC-PCR and FA.Experiments are now under way to define the toxoplasmic retinochoroiditis related genes by using cDNA microarray system