梗阻性黄疸大鼠肝细胞线粒体DNA损伤的定量研究

目的:对梗阻性黄疸大鼠肝细胞线粒体DNA损伤进行定量研究,探讨其与线粒体功能损伤的关系.方法:48只健康Wistar大鼠随机分为假手术组和梗阻性黄疸组,梗阻性黄疸组用胆总管双重结扎并切断制成胆道梗阻模型.Estabrook氧电极法测定线粒体呼吸控制率(RCR)和磷氧比值、Kimmich法测定肝组织内ATP含量以评价肝细胞线粒体功能,并利用荧光定量PCR方法,对各组大鼠肝细胞总mtDNA、缺失型mtDNA进行相对定量检测.结果:相对于假手术组,梗阻性黄疸组RCR、磷氧比值及肝组织ATP含量明显减少(P＜0.01),细胞内总mtDNA拷贝数减少(P＜0.01);假手术组内未检测出缺失型mtDNA,梗阻性黄疸组存在着缺失型mtDNA;随着梗阻时间的延长,梗阻性黄疸组RCR、磷氧比值及肝组织ATP含量逐步降低,总mtDAN拷贝数逐步减少(P＜0.01),缺失型mtDNA在总mtDNA中的百分比逐步增加(P＜0.01),其变化与线粒体功能损害相一致.结论:大鼠梗阻性黄疸肝细胞mtDNA损伤是导致线粒体功能损伤的重要因素.

Quantitative study of hepatocyte mtDNA damage in rats with obstructive jaundice

Objective:To quantitatively study the damage of hepatocyte mtDNA in rats with obstructive jaundice and to understand its relationship with the damage of mitochondrial function. Methods: Forty-eight Wistar rats were randomly divided into 2 groups, namely, sham-operation group and obstructive jaundice group (model group). The obstructive jaundice model was induced by double ligation and section of the common bile duct. The changes of mitochondrial function were routinely assessed; the whole mtDNA and deleted mtDNA in hepatocytes were determined by real-time fluorescent quantitative PCR.Results: Compared with sham-operation group, model group had reduced mitochondrial function and mtDNA copies (P<0.01). The deleted mtDNA was found in the model group but not in the sham-operation group. With the continuation of the obstruction time, mtDNA copies decreased in the model group and the percentage of deleted mtDNA in the whole mtDNA copies increased(P<0.01), which was consistant to the deterioration of mitochondrial function. Conclusion: The damage of hepatocyte mtDNA is an important factor for the damage of mitochondrial function in rats with obstructive jaundice.