Taguchi J, Murry C E, Herren B I, Pech M, Schwartz S M, Lindner V
First Department of Internal Medicine, University of Tokyo, Japan.
Am J Pathol. 1998 Apr;152(4):903-12.
Quantitation of mRNA expression by endothelial cells in vivo has been limited to larger animals from which sufficient amounts of RNA could be isolated for Northern blot analysis. In the present study, we established a technique to isolate endothelial RNA from rat aortas using en face preparations. This RNA was not contaminated with RNA from smooth muscle cells as demonstrated by the absence of smooth muscle alpha-actin RNA. Following lipopolysaccharide (LPS) administration to rats, quantitation of platelet-derived growth factor (PDGF) ligand and receptor mRNA expression was carried out by competitive reverse transcriptase-polymerase chain reaction and normalized to glyceraldehyde-3 phosphate dehydrogenase. The results of the competitive reverse transcriptase-polymerase chain reaction were compared with those obtained by en face in situ hybridization. Aortic endothelium showed a 140-fold increase in PDGF-A mRNA expression 4 hours after LPS injection. Expression levels of this growth factor declined to near base line levels within 36 hours of the LPS injection. A 52-fold increase in PDGF-B mRNA was seen at 12 hours after LPS injection but expression levels were approximately 300-fold lower than for PDGF-A. These data indicate that changes in PDGF expression by endothelium in vivo can greatly exceed those observed in cultured cells. This method should permit study of endothelial gene regulation in a variety of pathological conditions in vivo.
体内内皮细胞mRNA表达的定量分析仅限于较大的动物，因为只有从这些动物中才能分离出足够量的RNA用于Northern印迹分析。在本研究中，我们建立了一种使用主动脉整段标本从大鼠主动脉中分离内皮RNA的技术。如平滑肌α-肌动蛋白RNA的缺失所示，该RNA未被平滑肌细胞的RNA污染。给大鼠注射脂多糖（LPS）后，通过竞争性逆转录-聚合酶链反应对血小板衍生生长因子（PDGF）配体和受体mRNA的表达进行定量，并以甘油醛-3-磷酸脱氢酶进行标准化。将竞争性逆转录-聚合酶链反应的结果与通过主动脉整段原位杂交获得的结果进行比较。LPS注射后4小时，主动脉内皮的PDGF-A mRNA表达增加了140倍。在LPS注射后36小时内，这种生长因子的表达水平下降至接近基线水平。LPS注射后12小时，PDGF-B mRNA增加了52倍，但表达水平比PDGF-A低约300倍。这些数据表明，体内内皮细胞PDGF表达的变化可能大大超过在培养细胞中观察到的变化。该方法应有助于研究体内各种病理条件下的内皮基因调控。
