摘要目的 观察肝素对脂多糖(LPS)刺激肾脏微血管内皮细胞(RMVECs)形态学改变及一氧化氮(NO)和活性氧(ROS)表达的影响.方法 采用三步梯度筛网法原代培养大鼠RMVECs,取长势优异的3~4代细胞进行实验,并分为空白对照组、10 mg/L LPS处理组和2.5、5、10 kU/L肝素预处理组(LPS刺激前0.5 h给予相应剂量肝素干预).透射电镜下观察LPS刺激24 h细胞形态学改变;于LPS刺激5、15、30、45 min采用免疫荧光法检测RMVECs内ROS表达;采用硝酸还原酶法检测RMVECs内NO生成情况.结果 ① 空白对照组RMVECs细胞膜完整,细胞内线粒体和内质网清晰可见,核膜完整,核仁明显.LPS刺激24 h后,RMVECs细胞器空泡变性明显,以线粒体为主,且细胞膜产生大量微粒体.经10 kU/L肝素预处理后,细胞器空泡变性明显减轻,细胞膜形态稳定.② LPS刺激5 min即可在RMVECs细胞内检测到ROS表达和NO生成明显增加,并随时间延长逐渐增加,ROS表达30 min达峰值,NO生成45 min达峰值,与空白对照组同期比较差异均有统计学意义〔30 min ROS(平均荧光值):76.2±5.8比1.5±0.1,45 min NO(μmol/L):70.3±8.6比1.8±0.1,均P<0.01〕.随肝素剂量增加,预处理RMVECs后细胞内ROS表达及NO生成逐渐减少,以10 kU/L作用最明显,与LPS处理组同期比较差异均有统计学意义〔30 min ROS(平均荧光值):16.8±1.7比76.2±5.8,45 min NO(μmol/L):11.8±8.6比70.3±8.6,均P<0.01〕.结论 肝素可通过下调LPS诱导的NO、ROS表达和稳定细胞形态发挥保护肾脏内皮细胞的作用,以10 kU/L作用最明显.

abstractsObjective To observe the effect of heparin on the cellular morphology and the expressions of nitric oxide (NO) and reactive oxygen species (ROS) in renal microvascular endothelial cells (RMVECs) stimulated by lipopolysaccharide (LPS). Methods The three step gradient screen method was used to primarily culture rat RMVECs, and the 3rd and 4th generation cells with excellent growth were collected. The cells were divided into blank control group, 10 mg/L LPS treatment group and 2.5, 5, 10 kU/L heparin pretreatment groups (the corresponding dose of heparin was given 0.5 hour before LPS stimulation). The morphology of the cells at 24 hours after LPS stimulation was observed by transmission electron microscope, the expression of ROS in RMVECs was determined by immunofluorescence at 5, 15, 30, 45 minutes after LPS stimulation, and the expression of NO in RMVECs was determined by nitrate reductase method. Results ① In blank control group, the RMVECs membrane was intact, and the mitochondria and endoplasmic reticulum in cells were clearly visible. The nuclear membrane was complete, and nucleolus was obvious. Cell bubble deformation was obvious at 24 hours after LPS stimulation, especially in the mitochondria and cell membrane. After 10 kU/L heparin pretreatment, the vacuolar degeneration of organelles was significantly reduced, and the cell membrane morphology was stable. ② The increases in ROS and NO in RMVECs could be detected at 5 minutes after LPS stimulation, showed an increase tendency with time prolongation, ROS expression peaked at 30 minutes, NO expression peaked at 45 minutes, which showed significant differences as compared with those of blank control group [30-minute ROS (mean density): 76.2±5.8 vs. 1.5±0.1, 45-minute NO (μmol/L): 70.3±8.6 vs. 1.8±0.1, both P < 0.01]. The expression of ROS and NO production in RMVECs were significantly reduced by heparin, showed a decrease tendency with heparin dose elevation, and the most obvious effect was 10 kU/L of heparin, with significant difference as compared with those of LPS treatment group [30-minute ROS (mean density): 16.8±1.7 vs. 76.2±5.8, 45-minute NO (μmol/L): 11.8±8.6 vs. 70.3±8.6, both P < 0.01]. Conclusions Unfractionated heparin ameliorates LPS induced expressions of NO and ROS in RMVECs and protects the cell morphology. The effect of 10 kU/L heparin is most obvious.