球型脂联素对高糖环境下人脐静脉内皮细胞NO和DDAH-ADMA系统的影响

发布时间：2018-02-09 01:54

本文关键词： 高糖人脐静脉内皮细胞球型脂联素一氧化氮不对称二甲基精氨酸　出处：《南华大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：探讨球型脂联素（gad）对高糖环境下人脐静脉内皮细胞（HUVECs）NO及DDAH-ADMA系统的影响方法：体外培养HUVECs至对数生长期，gad(2.5ug/ml)处理高糖诱导不同时间（24h、48h）HUVECs，，不同浓度gad（0.1、0.5、2.5、12.5μg/ml）处理高糖诱导48h HUVECs，硝酸还原酶法测定培养液上清NO浓度；再将HUVECs随机分为正常对照组（5.5mmol/L葡萄糖）、高糖组（33.0mmol/L）、gad组（33.0mmol/L葡萄糖+2.5ug/ml gad），L-NAME组（33.0mmol/L葡萄糖+2.5μg/ml gad+100μmol/LL-NAME），硝酸还原酶法检测培养液上清NO浓度，化学比色法测定NOS活力，高效液相色谱法分析ADMA水平，根据ADMA的量反映DDAH活性，westernblot检测eNOS、P-eNOS的表达。结果： ①gad处理高糖诱导HUVECs不同时间（24h、48h），高糖作用24h，引起NO分泌增加，而作用48h，NO分泌显著减少（P0.05），与同一时间高糖组相比，gad组NO显著升高（P0.05）；②不同浓度gad（0.1、0.5、2.5、12.5μg/ml）处理高糖诱导48h HUVECs，NO水平先随gad浓度的增加而显著增加，但gad2.5μg/mL与12.5μg/mL相比，NO水平差异无显著性（P0.05）；③2.5μg/ml gad处理高糖诱导48h HUVECs，高糖组较对照组相比，NO含量显著降低，NOS活性及p-eNOS表达均减少（P0.05），而总eNOS表达无显著差异（P0.05）；④高糖组与正常对照组相比ADMA含量升高，DDAH活性降低，差异均有统计学意义（P0.05），2.5μg/mL gad处理后，与高糖组相比，NO含量显著升高，NOS活性显著增强，P-eNOS表达显著增加，总eNOS蛋白表达仍无统计学差异（P0.05）；ADMA水平gad组与高糖组相比降低，DDAH活性增加，差异均有统计学意义（P0.05），加入L-NAME100μmol/L，与gad组相比，NO含量显著减少，NOS活性减低，P-eNOS表达减少，差异有统计学差异（P0.05），总eNOS表达仍无差异（P0.05），ADMA水平升高，DDAH活性降低，差异均具有显著性（P0.05）。结论： 1、高糖环境下gad可逆转HUVECs合成NO的减少，并升高NOS活性，增加eNOS-Ser1177磷酸化，但对eNOS总蛋白表达并无影响。 2、高糖环境下gad可升高DDAH活性，降低ADMA水平，而这种作用可能依赖于gad升高NO水平，恢复NOS活性；同时，DDAM活性升高以及ADMA水平下降又可升高NO水平，增加NOS活性。
[Abstract]:Objective:. To investigate the effects of globular adiponectin on the nitric oxide (no) and DDAH-ADMA system of human umbilical vein endothelial cells (HUVECs) in high glucose environment. Methods:. HUVECs were incubated in vitro with HUVECs to logarithmic growth stage (2.5ugml) for 24 h / 48h after high glucose induction, and 0.5 渭 g / ml (0.5 渭 g / ml) for 48h after high glucose induction. Nitric acid reductase method was used to determine the concentration of nitric oxide (no) in the supernatant of cultured HUVECs. HUVECs was randomly divided into normal control group (5.5mmol / L glucose), high glucose group (33.0mmol / L) and high glucose group (33.0 mmol / L glucose 2.5ug-ml) -NAME group. Nitric acid reductase method was used to detect no concentration in the culture medium, and the activity of NOS was determined by chemical colorimetry. High performance liquid chromatography (HPLC) was used to analyze the level of ADMA and to detect the expression of eNOSn P-Enos by Western blot according to the activity of DDAH reflected by the quantity of ADMA. Results:. 1 g ad treatment with high glucose induced HUVECs at different time points for 24 h or 48 h, and high glucose for 24 h resulted in the increase of no secretion. Compared with the high glucose group at the same time, the no level in the Gad group was significantly higher than that in the high glucose group at the same time. The level of no increased significantly with the increase of the concentration of gad at 48h after treatment with 0.5 渭 g / ml of 0.5 渭 g / ml of gad. However, there was no significant difference in the level of no between gad2.5 渭 g / mL and 12.5 渭 g / mL in the treatment of HUVECs induced by high glucose for 48 h with 32.5 渭 g / ml gad. Compared with the control group, the content of nitric oxide monoxide synthase (no) and the expression of p-Enos in the high glucose group were significantly lower than those in the control group. There was no significant difference in the expression of total eNOS between the hyperglycemic group and the normal group. Compared with the control group, the ADMA content increased and the activity of DDAH decreased. After treatment with 2.5 渭 g / mL gad, the content of nitric oxide (no) increased significantly and the activity of nitric oxide synthase increased significantly (P 0.05). The expression of total eNOS protein was not significantly different between gad group and high glucose group, and the activity of gad group was significantly lower than that of high glucose group. Compared with gad group, the content of no decreased significantly and the expression of P-Enos decreased significantly (P 0.05). There was no significant difference in the expression of total eNOS. The level of total eNOS increased and the activity of DDAH decreased, and the difference was significant (P 0.05). Conclusion:. 1. Gad could reverse the decrease of no synthesis in HUVECs, increase the activity of NOS and increase the phosphorylation of eNOS-Ser1177, but had no effect on the expression of total eNOS protein. (2) gad could increase the activity of DDAH and decrease the level of ADMA in high glucose environment, which might depend on gad to increase the level of no and restore the activity of NOS, while the increase of activity of DDAM and the decrease of ADMA could increase the level of no and increase the activity of NOS.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【共引文献】