【作者】 闫骏；

【导师】 丛斌； 谷振勇；

【作者基本信息】 河北医科大学 ， 法医学， 2004， 硕士

【摘要】 目的：探讨八肽胆囊收缩素(CCK-8)对脂多糖（LPS）诱导人脐静脉内皮细胞株ECV-304内皮型一氧化氮合酶（endothelial nitric oxide synthase, eNOS）及诱导型一氧化氮合酶（inducible nitric oxide synthase, iNOS）表达的调节作用。方法：常规细胞培养；用LPS、CCK-8、LPS+CCK-8或溶剂分别孵育ECV-304细胞2h至24h不同时间，检测培养液上清中NO含量变化及细胞NOS活性变化；用免疫细胞化学方法及Western印迹方法检测细胞NOS蛋白表达量变化。结果：（1）LPS对ECV-304细胞NO含量、NOS活性及NOS蛋白表达的影响。①NO含量及NOS活性变化：1ug/ml LPS处理ECV-304 2h至24h，培养液上清中的NO含量随时间的延长而逐渐升高，与溶剂对照组相比均有明显差异；细胞NOS活性随时间延长而逐渐升高，与溶剂对照组相比均有明显增加。0.01、0.1和1ug/ml LPS处理ECV-304 2h和16h，NO含量随LPS浓度加大而升高，与溶剂对照组相比均有明显差异；细胞NOS活性随浓度的加大而逐渐升高，与溶剂对照组相比均有明显增加。②eNOS和iNOS蛋白表达变化：免疫细胞化学染色结果显示：eNOS在溶剂对照组ECV-304阳性表达主要分布于细胞膜、细胞浆中；LPS处理2h，细胞eNOS表达高于溶剂对照组，但细胞定位无变化。iNOS在溶剂对照组ECV-304细胞中几乎没有表达或表达量极少；LPS处理<WP=4>16h，iNOS在细胞浆中表达明显上调。Western印迹检测结果显示：eNOS蛋白在溶剂对照组中有表达；LPS处理2h，细胞eNOS蛋白表达量比溶剂对照组明显增加。iNOS在溶剂对照组中表达量极少；LPS处理16h，iNOS表达量明显高于溶剂对照组。（2）CCK-8对ECV-304细胞NO含量、NOS活性及NOS蛋白表达的影响。①NO含量及NOS活性变化：CCK-8处理细胞2h和16h，NO含量与溶剂对照组相比均无明显差异；细胞NOS活性与溶剂对照组相比均无明显变化。②eNOS和iNOS蛋白表达变化：免疫细胞化学染色结果显示： CCK-8处理2h，细胞eNOS表达及定位与溶剂对照组相比没有明显变化。CCK-8处理16h，iNOS表达及定位与溶剂对照组相比没有明显变化。Western印迹结果显示：eNOS在溶剂对照组中有表达；CCK-8处理2h，eNOS表达与溶剂对照组相比没有明显变化。CCK-8处理16h，细胞iNOS表达量与溶剂对照组相比略为上调。（3）CCK-8对LPS诱导下ECV-304 NO含量、NOS活性及NOS蛋白表达的影响。①NO含量及NOS活性变化：LPS和CCK-8处理细胞2h和16h，NO含量比LPS单独处理时相比明显降低，与溶剂对照组相比无明显变化。LPS和CCK-8处理细胞2h, 细胞NOS活性与LPS处理时相比无明显差异，与溶剂对照组相比均有明显增加。LPS和CCK-8处理细胞16h, 细胞NOS活性与LPS处理时相比有明显降低，与溶剂对照组相比有明显增加。②eNOS和iNOS蛋白表达变化：免疫细胞化学染色结果显示：LPS和CCK-8处理内皮细胞2h，eNOS表达与溶剂对照组相比明显增加，而与LPS处理时相比没有明显变化。LPS和CCK-8处理16h，iNOS表达比LPS处理时明显减少，但仍高于溶剂对<WP=5>照组。Western印迹结果：LPS和CCK-8处理细胞2h，eNOS表达与LPS处理2h相比没有明显变化，高于溶剂对照组。LPS和CCK处理16h，细胞iNOS表达比LPS处理时明显降低，但高于溶剂对照组。结论：LPS可诱导ECV-304 iNOS蛋白表达上调，NOS活性增加，NO生成增多；CCK-8对ECV-304 iNOS／eNOS表达，NOS活性和NO生成无明显影响，但CCK-8可抑制LPS诱导的iNOS蛋白表达上调，NOS活性增加及NO的生成增多。更多还原

【Abstract】 Objective: To investigate the regulatory effect of CCK-8 on LPS-induced inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in vascular endothelial cells.Methods: Human veinous endothelium line ECV-304 was cultured and incubated with LPS, CCK-8, LPS+CCK-8, or vehicle for different times. The supernatant of culture medium and cells were collected to measure NO content and NOS activity spectrophotometrically and to detect NOS expression by immunocytochemistry and Western blot.Results: (1)The effect of LPS on NO content, NOS activity and expression in ECV-304: Incubation of ECV-304 with 1ug/ml LPS resulted in a gradual increase of NO content in a time-dependent manner, which was markedly higher than that of vehicle group. The NOS activity also obviously enhanced in ECV-304 exposed to LPS compared with vehicle group. Incubation of ECV-304 with 0.01ug/ml, 0.1ug/ml and 1ug/ml LPS for 2h and 16h led to an obvious increase of NO content and NOS activity in a dose-dependent manner compared with vehicle group. The result of immunocytochemistry showed that eNOS expressed in the cytoplasm and membrane of ECV-304 in the <WP=7>vehicle group, and the eNOS expression enhanced in the LPS-stimulated ECV-304 for 2h without localization changes. The quantity of iNOS expression in the ECV-304 of the vehicle group is very little, which was obviously increased in LPS-stimulated cells for 16h. The Western blot assay showed the same results as above. (2) The effect of CCK-8 on NO content, NOS activity and expression in ECV-304: Incubation of ECV-304 with 10-6mol/L、10-7mol/L and 10-8mol/L CCK-8 for 2h and 16h led to no difference compared with vehicle group on NO content and NOS. The result of immunocytochemistry showed that the eNOS expression has no alteration in the CCK-8-stimulated ECV-304 for 2h without localization changes compared with the vehicle group. The quantity of iNOS expression in the ECV-304 of vehicle group is very little, which was not different from CCK-8-stimulated cells for 16h. The Western blot assay showed the same results as above. (3) The effect of CCK-8 on NO content, NOS activity and expression in ECV-304 exposed to 0.1ug/ml LPS: Incubation of ECV-304 exposed to 0.1ug/ml LPS with 10-6mol/L、10-7mol/L and 10-8mol/L CCK-8 led to an obvious decrease of NO content compared with LPS-stimulated group. And it was not different from vehicle group. The NOS activity in CCK-8 and LPS group for 2h was not different from LPS-stimulated group and higher than that of vehicle group. But the NOS activity in CCK-8 and LPS group for 16h was lower than that of LPS-stimulated group and higher than that of vehicle group. The result of <WP=8>immunocytochemistry showed that the expression of eNOS in CCK and LPS group for 2h was not different from that of vehicle group. The quantity of iNOS expression was obviously increased in CCK-8 and LPS group compared with the vehicle group. The Western blot assay showed the same result as above.Conclusion: LPS could induce the upregulation of NOS and increased NOS activity and NO contents. CCK-8 had no effect on iNOS/eNOS expression, NOS activity and NO contents in ECV-304, but it could inhibit LPS-induced the upregulation of iNOS expression, increased NOS activity and NO contents.更多还原