Sridulyakul P, Chakraphan D, Bhattarakosol P, Patumraj S
Field of Medical Science, Graduate School, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.
Clin Hemorheol Microcirc. 2003;29(3-4):423-8.
To compare the level of endothelial nitric oxide synthase (eNOS) expression produced in heart and lung vascular tissue, the protein content was determined using Western blot analysis with the enhancement of image processing. Heart and lung extracts from 12 and 24 weeks from control (CON) and streptozotocin-induced diabetic (DM) rats were collected for Western blot analysis. Using monoclonal antibody against rat eNOS protein (140 kDa), the eNOS-protein bands were detected with enhanced chemiluminescence (ECL; Amersham) and exposured to film (Hyperfilm-ECL; Amersham). Images of eNOS bands on each film were then scanned and saved to digital files. Using Global Lab Image software, the number of pixels in each digital file was counted and calibrated for eNOS-protein content. For the CON and DM groups, the mean values of eNOS-protein contents were calculated and expressed as a percentage of total protein content, 5 micrograms. It was found that the eNOS level in DM hearts was significantly decreased, as compared to age-matched CON hearts. On the other hand, eNOS levels in DM lungs was increased, compared to CON lungs. Therefore, it may be concluded that high, not low, flow-mediated eNOS expression is a good measure of hyperglycemic-induced endothelial dysfunction.
为了比较心脏和肺血管组织中内皮型一氧化氮合酶（eNOS）的表达水平，采用增强图像处理的蛋白质印迹分析法测定蛋白质含量。收集来自对照（CON）大鼠和链脲佐菌素诱导的糖尿病（DM）大鼠12周和24周时的心脏和肺提取物用于蛋白质印迹分析。使用抗大鼠eNOS蛋白（140 kDa）的单克隆抗体，通过增强化学发光法（ECL；Amersham）检测eNOS蛋白条带，并将其曝光于胶片（Hyperfilm-ECL；Amersham）。然后扫描每张胶片上eNOS条带的图像并保存为数字文件。使用Global Lab Image软件，对每个数字文件中的像素数量进行计数并校准以得出eNOS蛋白含量。对于CON组和DM组，计算eNOS蛋白含量的平均值，并表示为总蛋白含量（5微克）的百分比。结果发现，与年龄匹配的CON组心脏相比，DM组心脏中的eNOS水平显著降低。另一方面，与CON组肺相比，DM组肺中的eNOS水平升高。因此，可以得出结论，高流量而非低流量介导的eNOS表达是高血糖诱导的内皮功能障碍的良好指标。
