Lei X, Buja L M
Department of Pathology, University of Texas Medical School, Houston 77030, USA.
Lab Invest. 1996 Jan;74(1):136-45.
Tumor necrosis factor-alpha (TNF-alpha), produced by activated monocytes and other cells, has been proposed as a mediator of importance in atherosclerosis. In this study, we use a newly developed technique, quantitative reverse transcription-polymerase chain reaction (RT-PCR), to measure the mRNA levels of TNF-alpha in arteries of Watanabe Heritable Hyperlipidemic (WHHL) rabbits in relation to the progression of atherosclerosis. Co-amplification of known amounts of TNF-alpha RNA and TNF-alpha internal control RNA indicated that the quantitative RT-PCR method was quite reliable, with a < 5% difference between TNF-alpha mRNA levels deduced from the standard curve and actually loaded TNF-alpha mRNA. As another control, TNF-alpha mRNA levels in lipopolysaccharide-induced (LPS-induced) and uninduced monocytes were measured, and a 9.3-fold increase in the TNF-alpha mRNA levels was observed in LPS-induced monocytes. TNF-alpha mRNA levels in the aortic arches of healthy New Zealand White (NZW) rabbits was 0.0112 -/+ 0.0016 (SD) pg per ng tissue RNA. TNF-alpha mRNA levels in the aortic arches and descending thoracic aortas of 6-month-old WHHL rabbits were 0.0273 -/+ 0.0066 pg and 0.0176 -/+ 0.0013 pg per ng tissue RNA, respectively. TNF-alpha mRNA levels in the aortic arches and descending thoracic aortas of 18-month-old WHHL rabbits were much higher than in those of 6-month-old WHHL rabbits, with values of 0.2107 -/+ 0.0205 pg and 0.1043 -/+ 0.0196 pg per ng tissue RNA, respectively. These findings indicate that: a) Quantitative RT-PCR can be used to measure levels of small abundance RNA in normal and diseased tissues accurately; b) no significant increase in TNF-alpha mRNA levels was observed in the aortic arches and descending thoracic aortas of 6-month-old WHHL rabbits compared with those of NZW rabbits, although they had multiple raised intimal lesions; and c) a significantly elevated total tissue level of TNF-alpha mRNA is demonstrable late in the course of atherosclerosis in 18-month-old WHHL rabbits (Bonferroni method). These findings suggest that TNF-alpha might play an important role in the progression of advanced atherosclerosis, although total tissue levels of TNF-alpha mRNA are not unequivocally elevated earlier in the course of the disease.
肿瘤坏死因子-α（TNF-α）由活化的单核细胞和其他细胞产生，被认为是动脉粥样硬化中一种重要的介质。在本研究中，我们使用一种新开发的技术，即定量逆转录-聚合酶链反应（RT-PCR），来测量与动脉粥样硬化进展相关的渡边遗传性高脂血症（WHHL）兔动脉中TNF-α的mRNA水平。已知量的TNF-α RNA和TNF-α内部对照RNA的共扩增表明，定量RT-PCR方法相当可靠，从标准曲线推导的TNF-α mRNA水平与实际加载的TNF-α mRNA之间的差异<5%。作为另一个对照，测量了脂多糖诱导（LPS诱导）和未诱导的单核细胞中TNF-α mRNA水平，观察到LPS诱导的单核细胞中TNF-α mRNA水平增加了9.3倍。健康新西兰白兔（NZW）主动脉弓中TNF-α mRNA水平为每纳克组织RNA 0.0112±0.0016（标准差）皮克。6月龄WHHL兔主动脉弓和胸降主动脉中TNF-α mRNA水平分别为每纳克组织RNA 0.0273±0.0066皮克和0.0176±0.0013皮克。18月龄WHHL兔主动脉弓和胸降主动脉中TNF-α mRNA水平远高于6月龄WHHL兔，分别为每纳克组织RNA 0.2107±0.0205皮克和0.1043±0.0196皮克。这些发现表明：a）定量RT-PCR可用于准确测量正常和患病组织中小丰度RNA的水平；b）6月龄WHHL兔主动脉弓和胸降主动脉中TNF-α mRNA水平与NZW兔相比未观察到显著增加，尽管它们有多个内膜病变；c）在18月龄WHHL兔动脉粥样硬化病程后期可证明TNF-α mRNA的总组织水平显著升高（Bonferroni法）。这些发现表明，尽管在疾病病程早期TNF-α mRNA的总组织水平并未明确升高，但TNF-α可能在晚期动脉粥样硬化进展中起重要作用。
