AP-1对氧化LDL诱导系膜细胞TGFβ1转录调控的研究

The transforming growth factor(TGF-beta1) is an important factor in renal diseases. And in normal physiological metabolism it also regulate the production and degradation of excellular matrix. If it was inhibited, normal metabolism will be affected. Different stimulus induced overexpression of TGF-beta-1 by different pathways. So, we explored its function on transcription level. Oxidized Low Density Lipoprotein(Ox-LDL)-induced overproduction of the prosclerotic cytokine transforming growth factor-beta1 has been implicated in the pathogensis of renal fibrosis and sclerosis. Because Ox-LDL increase TGF-beta1 mRNA levels in rat mesangial cells, our investigation was designed to characterize these effects on the rat TGF-beta1 promoter activity. We constructed a luciferase-reporting system using the promoter of TGF beta 1(from -1550 to +57bp) and perform various deletions. Then we transfected these constructs to rat mesangial cells. The TGF-beta1 promoter activity enhanced induced by Ox-LDl. we found that two regions responsible for the induction; One is a negative regulatory region (-422 to -629) which represses the transcription of TGF-beta1 gene , the other is a positive regulatory region (-845 to -1550) which enhance the transcription unit efficiently. We mutated the site of cis-element sequence. After site-directed-mutation of cis-element we found the promoter activity exchanged. Then we presumed some transcription factor were probably involved in the regulation of TGF-beta1 transcription. Gel shift assay indicated that AP-1 and NF-kappaB was involved in transcription of TGF-beta1 induced by Ox-LDL. And super gel shift assay was performed, the c-jun of AP-1 and p50,p65 of NF-kappaB were significantly activated. Then many protein kinases were used, we found Ox-LDL doesn't stimulate protein expression of MARK subfamilies and phosphorylation of p44/42 MARK in mesangial cells, but significantly increased the phosphorylation of JNK1/SAPK and p38 MARK. Finally AP-1 and NF-kappaB decoy oligodeoxynucleotides were used to treat the overexpression of TGF-beta1 induced by Ox-LDL. The induction were abolished, without affecting the proliferation and growth of the rat mesangial cells.

在培养的人系膜细胞中加入氧化LDL，导入HVJ脂质体AP-1诱捕寡脱氧核苷酸，研究AP-1对TGF 1转录的作用。构建TGF 1启动子CAT报告基因表达载体并转染细胞，比较AP-1成分和蛋白激酶活性改变并观察蛋白激酶抑制剂，抗氧化剂后的变化，探索AP-1对氧化LDL诱导TGF1转录调控机制和信号传递通路。探讨控制TGF 1过度表达和脂质肾损害的治疗方法。..

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