mtTFA、NRF-1对冷保存-再灌注肝移植大鼠线粒体DNA ATPase6基因表达的调节

Effects of mitochondrial transcription factor A and nuclear respiratory factor-1 on the expression of mitochondrial DNA ATPase6 genes during cold preservation and reperfusion injury in rat orthotopic liver transplantation

目的:研究线粒体DNA上游调节基因mtTFA和NRF-1对冷保存肝移植大鼠肝细胞线粒体DNA ATPase6基因表达的影响,探讨mtTFA和NRF-1对冷保存肝移植大鼠肝细胞线粒体DNA调节作用及其机制.方法:Wistar大鼠186只,采用改良"二袖套法"制作大鼠肝移植模型,动物随机分为A组:冷保存30min;B组:冷保存6h;C组:冷保存12h;和D组(假手术对照组),分别于制模后于12h、24h、3d、5d、7d采集标本,保证每时相点存活大鼠6只.观察各组大鼠肝脏ATP含量.采用RT-PCR方法检测mtDNA ATPase6 mRNA、mtTFA和NRF-1 mRNA的表达变化.结果:冷保存再灌注后早期(12h),A、B、C三组mtTFA mRNA表达降低,与A、B组相比,C组最为显著(0.57±0.05vs0.87±0.11,0.69±0.10,P<0.05),NRF-1 mRNA表达变化与mtTFA mRNA相一致.24h后各组mtTFA及NRF-1 mRNA表达开始升高,ATPase6表达和肝组织ATP含量也升高,并且升高趋势与mtTFA及NRF-1 mRNA表达增高基本一致.结论:mtTFA和NRF-1可能通过基因转录调节ATPase6基因表达,改变线粒体呼吸链转运电子、合成ATP的能力.

AIM： To observe the effects of mitochondrial transcription factor A （mtTFA） and nuclear respiratory factor-1 （NRF-1） on the expression of mitochondrial ATP6 genes during cold preservation and reperfusion injury in rats receiving orthotopic liver transplantation. METHODS： Orthotopic liver transplantation was performed in Wistar rats using the cuff technique as described by Kamada with modifications. A total of 186 rats were randomly divided into 4 groups, named as group A （30 min of cold preservation）, group B （6 h of cold preservation）, group C （12 h of cold preservation） and group D （sham operation）. Hepatic samples were collected at 12 h, 24 h and on the 3^rd, 5^th, 7^th day after operation. The ATP levels were observed in each group. The expression levels of NRF-1, mtTFA and mtDNA encoding ATPase6 mRNA were determined by reverse transcription-polymerase chain reaction （RT-PCR）. RESULTS： The expression of mtTFA decreased in groups A, B and C 12 h after operation, and it was lower in group C and groups A and B （0.57 ± 0.05 vs 0.87 ± 0.11, 0.69 ± 0.10, P 〈 0.05）. The expression change of NRF-1 mRNA was consistent with that of mtTFA. After 24 h, the expression levels of mtTFA and NRF-1 mRNA started to increase, and the expression of ATPase6 mRNA and ATP in hepatic tissues were in accordance with mtTFA and NRF-1 mRNA. CONCLUSION： mtTFA and NRF-1 increase the expression of ATPase6 mRNA, suggesting mtTFA and NRF-1 may be important factors in controling ATPase6 mRNA transcription.