Proteomic Analyses of Lung Lysates from Short-Term Exposure of Fischer 344 Rats to Cigarette Smoke
Proteomic Analyses of Lung Lysates from Short-Term Exposure of Fischer 344 Rats to Cigarette Smoke
复制标题
DOI：
10.1021/pr200345y
复制
发表时间：
2011-08-01
影响因子：
4.4
通讯作者：
Pelech, Steven
中科院分区：
文献类型：
--
作者：
Carter, Charleata A.;Misra, Manoj;Pelech, Steven
在Fischer 344大鼠中进行了为期5天的短期主流香烟烟雾暴露研究，以确定肺蛋白的变化。每组10只5周龄的雄性和雌性大鼠被分为四个暴露组。动物们接受了仅用鼻子过滤的空气（空气控制）或75、200或400 mg总颗粒物(TPM)/m(3)的稀释香烟烟雾。每天暴露3小时，连续5天。每只动物的一个肺被冷冻在液氮中，并进行蛋白质组学分析。使用抗体微阵列技术筛选650种抗体，检测信号蛋白水平和磷酸化的变化，然后通过免疫印迹法评估超过100种顶级蛋白。使用反向裂解物微阵列进一步评估顶部烟雾改变蛋白。主要蛋白变化在Western blots上显示中至强条带，取决于剂量和性别，包括蛋白丝氨酸激酶（Cot/Tpl2、ERK1/2、GSK3 α / β、MEK6、PKC α / γ、RSK1）、蛋白磷酸酶（PP4/A'2、PP1C β）和其他蛋白（caspase 5、CRMP2、Hsc70、Hsp60、Rac1和STAT2）。雌性暴露于75 mg TPM/m(3)时，雄性暴露于200 mg TPM/m3时，变化最为明显。烟雾改变的蛋白质调节细胞凋亡、应激反应、细胞结构和炎症。鉴定蛋白的变化可作为肺损伤的早期指标。
A short-term 5 day mainstream cigarette smoke exposure study was conducted in Fischer 344 rats to identify changes in lung proteins. Groups of 10 male and female rats at 5 weeks of age were assigned to one of four exposure groups. Animals received either nose-only filtered air (Air Control) or 75, 200, or 400 mg total particulate matter (TPM)/m(3) of diluted cigarette smoke. Exposures were conducted for 3 h per day, for 5 consecutive days. One lung per animal was frozen in liquid nitrogen and processed for proteomic analyses. Lung lysates from control verses treated animals were screened with 650 antibodies for changes in signaling protein levels and phosphorylation using antibody microarray technology, and then over 100 of the top protein hits were assessed by immunoblotting. The top smoke-altered proteins were further evaluated using reverse lysate rnicroarrays. Major protein changes showed medium to strong bands on Western blots, depended on dose and gender, and included protein-serine kinases (Cot/Tpl2, ERK1/2, GSK3 alpha/beta, MEK6, PKC alpha/gamma, RSK1), protein phosphatases (PP4/A'2, PP1C beta), and other proteins (caspase 5, CRMP2, Hsc70, Hsp60, Rac1 and STAT2). The most pronounced changes occurred with 75 mg TPM/m(3) exposed females and 200 mg TPM/m3 exposed males. Smoke-altered proteins regulate apoptosis, stress response, cell structure, and inflammation. Changes in identified proteins may serve as early indicators of lung damage.