血管平滑肌细胞快速牵张损伤后细胞缝隙连接功能的变化.DOCVIP

临床医学论文-血管平滑肌细胞快速牵张损伤后细胞缝隙连接功能的变化 ??????????????? 作者：徐锡金，霍霞，李燕，Margaret A? ［摘要］目的：探讨牵张损伤时血管平滑肌细胞(A7r5)缝隙连接功能的改变及其影响因素。方法：采用细胞牵张损伤装置建立A7r5细胞损伤模型，通过细胞培养、荧光显微镜、共聚焦激光扫描显微镜荧光漂白后恢复技术检测对照组，轻度、中度和重度牵张损伤组细胞活性(Hoechst 33342和propidium iodide染色)、细胞内钙(Fluo_4/AM染色)、细胞内氧自由基(H2DCFDA染色)和细胞间通讯功能(5_CFDA染色)的变化。结果：随着细胞牵张损伤的加重，细胞活性降低、细胞内钙及细胞内氧自由基升高、细胞间通讯功能下调。含钙缓冲液和细胞间通道阻断剂甘珀酸可使细胞通讯功能进一步下调，而Ca2＋鳌合剂――乙二醇四乙酸、氧自由基清除剂――超氧化物歧化酶可使细胞通讯功能上调。结论：牵张损伤可通过细胞内钙超载和活性氧的增多来降低平滑肌细胞缝隙连接的细胞通讯功能。 　　［关键词］平滑肌细胞(A7r5)；细胞损伤；缝隙连接 　　Abstract］Objective: To estimate the role and affected mechanism of gap junctions during cell injury. Methods: A7r5 cells were seeded onto six_well FlexPlates that had a silastic bottom and provided the ability to deform and injure the cells. Mild, moderate and severe groups of rapid stretch injury(RSI, 55, 65, 75 mm deformation)were produced u_sing a model 94A cell injury controller. Cell injury was defined as the percent of Hoechst 33343 stained cells that stained with propidium iodide(PI). Gap junction communication was assayed using fluorescence recovery after photobleaching(FRAP). Cells were loaded with 5_carboxyfluorescein diacetate and intracellular fluorescence was measured using confocal laser scanning microscopy(CLSM). FRAP was expressed as percent of baseline fluorescence. Intracellular Ca2+(［Ca2＋］i)and reactive oxygen species(ROS)were imaged under CLSM using the fluorescent dyes fluo_4 AM and H2DCFDA/AM, respectively. Results: RSI produced level dependent increased in PI_positive cells, ［Ca2＋］i and ROS, but decreased in gap junctional communication. DPBS with calcium and gap junction blocker Carbenoxalone could further decrease gap junction communication; whereas antioxidant SOD or the calcium chelator EGTA could increase gap junction communication. Conclusion: These results suggest that increases in ［Ca2＋］i and ROS contribute to cell injury and impaired gap junctional communication after RSI in A7r5 cells in vitro. 　　［Key Words］A7r5 Cell; cell injury; gap junction 　　创伤性脑损伤(