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目的观察大鼠肾小球膜细胞137Cs照后PAI1基因的表达情况,以及PPARγ配体Pioglitazone对照射诱导的PAI1表达的影响。方法10Gyγ线照射±10mmol/LPioglitazone处理大鼠肾小球膜细胞,用RTPCR方法证明PPARγ基因表达,用Northernblot、Westernblot方法观察PAI1基因表达,用Gelshift方法观察AP1基因的活性变化(由于PAI1基因启动子区存在AP1结合位点,加之AP1介导某些炎性因子的产生且AP1是照射诱导的早期表达基因之一,因此观察照射在大鼠肾小球膜细胞能否诱导AP1活性的升高,并进一步观察Pioglitazone对照后AP1活性的影响)。结果RTPCR方法证明了大鼠肾小球膜细胞表达PPARγ基因。10Gy照后Northernblot结果显示照后24hPAI1mRNA的表达增加,不同剂量照后24hPAI1mRNA的高表达呈照射剂量依赖性。10mmol/LPioglitazone+10Gy照后24h的Northernblot和48h的Westernblot结果表明,Pioglitazone使照射诱导的PAI1基因高表达在mRNA水平和蛋白水平均明显降低。Gelshift结果发现放疗后2hAP1活性开始升高,4h最高,加入Pioglitazone后明显被抑制。结论PPARγ配体Pioglitazone使照射诱导的肾小球膜细胞PAI1基因高表达降低,其机制可能是通过降低AP1活性来调节PAI1的表达。Pioglitazone可能具有预防或降低放射性肾损伤的作用,这将有待于动物实验的进一步证实。
Objective To investigate the expression of PAI1 gene in rat mesangial cells (137Cs) and the effect of PPARγ ligand Pioglitazone on PAI1 expression. Methods The rat glomerular mesangial cells were treated with 10 Gy γ-ray (± 10 mmol / L LPioglitazone). The expression of PPARγ gene was confirmed by RTPCR. The expression of PAI1 gene was observed by Northern blot and Western blot. The changes of AP1 gene activity were observed by Gelshift (PAI1 gene promoter AP1 binding sites exist in the region, combined with the AP1-mediated production of certain inflammatory factors and AP1 is induced by irradiation one of the early expression of genes, so observe the irradiation of rat mesangial cells can induce AP1 activity increased, And further observe the effect of AP1 activity after Pioglitazone control). Results The RTPCR method demonstrated that rat mesangial cells expressed PPARγ gene. The results of Northern blot showed that the expression ofPAI1mRNA increased at24h after irradiation, and the high expression ofPAI1mRNA at24h after irradiation was dose dependent. Western blot analysis of Northernblot and 48h after 24h with 10mmol / LPioglitazone + 10Gy showed that Pioglitazone significantly decreased the expression of PAI1 gene at mRNA and protein levels. Gelshift results showed that 2h after radiotherapy, the activity of PAP1 began to increase, reaching the highest at 4h, which was obviously inhibited after adding Pioglitazone. Conclusions PPARγ ligand Pioglitazone can reduce PAI1 expression in mesangial cells induced by irradiation. The mechanism may be that PAI1 is regulated by decreasing AP1 activity. Pioglitazone may have the effect of preventing or reducing radiation-induced renal damage, which will need to be further confirmed in animal experiments.