氯化钠抑制内皮型一氧化氮合成酶活性及其机制探讨
发布时间：2018-08-04 21:49
【摘要】： 目的 通过内皮型一氧化氮合成酶(endothelial nitric oxide synthase, eNOS)活性分析证明:微量氯化钠(sodium chloride, NaCl)浓度(5～20mmol/L)的升高能够显著抑制eNOS活性,使一氧化氮(nitric oxide, NO)合成减少。通过乙酰胆碱(acetylcholine, Ach)和钙离子载体A23187(eNOS激活剂)做对照,进一步探讨NaCl抑制eNOS活性的分子机制。 方法 1.细胞实验:应用原代小牛动脉内皮细胞(primary bovine endothelial cells, BA EC)及表达eNOS或者vector的中国仓鼠卵巢细胞(Chinese hamster ovary cells, CHO)进行Western Blot及eNOS活性分析。 2.排除实验干扰因素:应用等毫摩尔甘露醇代替NaCl培养的BAEC细胞系进行eNOS活性分析、应用BAEC细胞系进行WST染色及3H-L-精氨酸摄取能力实验,排除渗透压、细胞活性改变及3H-L-精氨酸摄取能力改变对实验结果的影响。 3.动脉内皮细胞再生分析:应用eNOS抑制剂NG-硝基-L-精氨酸甲酯(NG-Nitro- L- arginine Methyl Ester, L-NAME)和NaCl分别培养小鼠主动脉环,观察NaCl对小鼠动脉内皮细胞再生的影响。 4.延伸实验:分别用Ach、Ach加NaCl、A23187、A23187加NaCl培养的细胞分析eNOS活性,进一步探讨NaCl抑制eNOS活性的分子机制。 结果 1.通过BAEC、CHO-eNOS、CHO-vector细胞系进行Western Blot显示:CHO-vector细胞系不表达eNOS蛋白,而BAEC和CHO-eNOS细胞系表达eNOS蛋白,说明可以用后两种细胞系进行eNOS活性分析。 2.通过应用表达eNOS蛋白的BAEC细胞系和CHO-eNOS细胞系进行eNOS活性分析发现:培养细胞所用的NaCl浓度越高,eNOS活性越低(P0.0001),NaCl对eNOS活性的抑制呈剂量依赖关系。实验中,NaCl孵育细胞15min就能显著抑制eNOS活性。 3.通过应用等毫摩尔甘露醇代替不同浓度NaCl培养细胞进行eNOS活性分析,证明eNOS活性的抑制不是由于加入NaCl后渗透压改变引起的(P0.05);通过对加NaCl(137～157mmol/L)培养的BAEC细胞系进行WST染色,发现NaCl培养组细胞存活率与PBS培养组相比无显著性差异(P0.05);通过应用BAEC细胞系进行3H-L-精氨酸摄取能力分析(3H-L-Arginine uptake assay),证明不同浓度NaCl对L-精氨酸进入细胞的量无显著性影响(P0.05)。 4.通过进行动脉内皮细胞再生实验,发现培养液中加入eNOS抑制剂L-NAME后,动脉内皮细胞生长非常缓慢。培养液中加入NaCl的二组细胞同样生长缓慢,尤其是加入的NaCl浓度为20mmol/L的一组,内皮细胞生长状况近似于L-NAME组。 5.NaCl抑制eNOS活性的分子机制的研究:发现eNOS激活剂Ach和A23187培养的细胞eNOS活性增强,但是同时加入NaCl培养细胞后,增强的eNOS活性被抑制,并且NaCl浓度越高,对eNOS活性的抑制越显著(P0.05)。 结论 1.微量NaCl浓度的升高(5～20mmol/L)就能显著抑制eNOS活性,这种抑制发生的非常迅速(15min内)并且呈剂量依赖关系。eNOS活性降低使NO合成减少,血管收缩,血压升高。 2. NaCl对eNOS活性的抑制可能是通过影响Ca2+入胞实现的。
[Abstract]:Objective to analyze the activity of endothelial nitric oxide synthase (endothelial nitric oxide synthase, eNOS) and to prove that the increase of (sodium chloride, NaCl) concentration (5~20mmol/L) can significantly inhibit the activity of eNOS and decrease the synthesis of (nitric oxide, NO). By using acetylcholine (acetylcholine, Ach) and calcium carrier A23187 (eNOS activator) as controls, the molecular mechanism of NaCl inhibiting eNOS activity was further explored. Method 1. Cell experiment: the activities of Western Blot and eNOS were analyzed by primary bovine artery endothelial cells (primary bovine endothelial cells, BA EC) and Chinese hamster ovarian cells (Chinese hamster ovary cells, CHO) expressing eNOS or vector. 2. Eliminating experimental interference factors: eNOS activity of BAEC cell line cultured with isomolor mannitol instead of NaCl was analyzed, WST staining and 3H-L- arginine uptake ability of BAEC cell line were used to remove osmotic pressure. Effects of changes in cell activity and 3H-L-arginine uptake on the results. 3. 3. Arterial endothelial cell regeneration analysis: eNOS inhibitor NG-nitro-L-arginine methyl ester (NG-Nitro-L- arginine Methyl Ester, L-NAME) and NaCl were used to culture mouse aortic rings, and the effects of NaCl on the regeneration of mouse arterial endothelial cells were observed. 4. Extension experiment: the eNOS activity was analyzed by using the cells cultured with A23187A23187 and NaCl, respectively, and the molecular mechanism of the inhibition of eNOS activity by NaCl was further discussed. Result 1. Western Blot analysis showed that eNOS protein was not expressed in BAEC and CHO-eNOS cell lines, suggesting that the latter two cell lines could be used for eNOS activity analysis. 2. The eNOS activity of BAEC and CHO-eNOS cell lines expressing eNOS protein was analyzed in a dose-dependent manner. It was found that the higher the NaCl concentration was, the lower the Enos activity (P0.0001) could inhibit eNOS activity in a dose-dependent manner. In the experiment, 15min could significantly inhibit the activity of eNOS. By using isomolor mannitol instead of different concentration of NaCl to analyze the eNOS activity, it was proved that the inhibition of eNOS activity was not caused by the change of osmotic pressure after adding NaCl (P0.05), and the BAEC cell line cultured with NaCl (137~157mmol/L) was stained with WST. It was found that there was no significant difference in cell survival rate between NaCl culture group and PBS culture group (P0.05), and the 3H-LL-arginine uptake ability of BAEC cell line was analyzed by using BAEC cell line (3H-L-Arginine uptake assay), proved that there was no significant difference in the amount of L-arginine entering cells with different concentrations of NaCl). Effects (P0.05). 4. Through the experiment of arterial endothelial cell regeneration, it was found that the growth of arterial endothelial cells was very slow after the addition of eNOS inhibitor L-NAME in the culture medium. The two groups of cells added with NaCl in the culture medium also grew slowly, especially when the concentration of NaCl was 20mmol/L. The growth status of endothelial cells was similar to that of L-NAME group. The molecular mechanism of 5.NaCl inhibiting eNOS activity was studied. It was found that the eNOS activity of eNOS activator Ach and A23187 cells was increased, but the cells were cultured with NaCl. The enhanced eNOS activity was inhibited, and the higher the concentration of NaCl was, the more significant the inhibition of eNOS activity was (P0.05). Conclusion 1. The increase of NaCl concentration (5~20mmol/L) significantly inhibited the activity of eNOS. The inhibition occurred very quickly (within 15min) and decreased the activity of Enos in a dose-dependent manner, resulting in the reduction of no synthesis, vasoconstriction and elevated blood pressure. The inhibition of eNOS activity by NaCl may be achieved by affecting Ca2 entry.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363
本文编号：2165270
[Abstract]:Objective to analyze the activity of endothelial nitric oxide synthase (endothelial nitric oxide synthase, eNOS) and to prove that the increase of (sodium chloride, NaCl) concentration (5~20mmol/L) can significantly inhibit the activity of eNOS and decrease the synthesis of (nitric oxide, NO). By using acetylcholine (acetylcholine, Ach) and calcium carrier A23187 (eNOS activator) as controls, the molecular mechanism of NaCl inhibiting eNOS activity was further explored. Method 1. Cell experiment: the activities of Western Blot and eNOS were analyzed by primary bovine artery endothelial cells (primary bovine endothelial cells, BA EC) and Chinese hamster ovarian cells (Chinese hamster ovary cells, CHO) expressing eNOS or vector. 2. Eliminating experimental interference factors: eNOS activity of BAEC cell line cultured with isomolor mannitol instead of NaCl was analyzed, WST staining and 3H-L- arginine uptake ability of BAEC cell line were used to remove osmotic pressure. Effects of changes in cell activity and 3H-L-arginine uptake on the results. 3. 3. Arterial endothelial cell regeneration analysis: eNOS inhibitor NG-nitro-L-arginine methyl ester (NG-Nitro-L- arginine Methyl Ester, L-NAME) and NaCl were used to culture mouse aortic rings, and the effects of NaCl on the regeneration of mouse arterial endothelial cells were observed. 4. Extension experiment: the eNOS activity was analyzed by using the cells cultured with A23187A23187 and NaCl, respectively, and the molecular mechanism of the inhibition of eNOS activity by NaCl was further discussed. Result 1. Western Blot analysis showed that eNOS protein was not expressed in BAEC and CHO-eNOS cell lines, suggesting that the latter two cell lines could be used for eNOS activity analysis. 2. The eNOS activity of BAEC and CHO-eNOS cell lines expressing eNOS protein was analyzed in a dose-dependent manner. It was found that the higher the NaCl concentration was, the lower the Enos activity (P0.0001) could inhibit eNOS activity in a dose-dependent manner. In the experiment, 15min could significantly inhibit the activity of eNOS. By using isomolor mannitol instead of different concentration of NaCl to analyze the eNOS activity, it was proved that the inhibition of eNOS activity was not caused by the change of osmotic pressure after adding NaCl (P0.05), and the BAEC cell line cultured with NaCl (137~157mmol/L) was stained with WST. It was found that there was no significant difference in cell survival rate between NaCl culture group and PBS culture group (P0.05), and the 3H-LL-arginine uptake ability of BAEC cell line was analyzed by using BAEC cell line (3H-L-Arginine uptake assay), proved that there was no significant difference in the amount of L-arginine entering cells with different concentrations of NaCl). Effects (P0.05). 4. Through the experiment of arterial endothelial cell regeneration, it was found that the growth of arterial endothelial cells was very slow after the addition of eNOS inhibitor L-NAME in the culture medium. The two groups of cells added with NaCl in the culture medium also grew slowly, especially when the concentration of NaCl was 20mmol/L. The growth status of endothelial cells was similar to that of L-NAME group. The molecular mechanism of 5.NaCl inhibiting eNOS activity was studied. It was found that the eNOS activity of eNOS activator Ach and A23187 cells was increased, but the cells were cultured with NaCl. The enhanced eNOS activity was inhibited, and the higher the concentration of NaCl was, the more significant the inhibition of eNOS activity was (P0.05). Conclusion 1. The increase of NaCl concentration (5~20mmol/L) significantly inhibited the activity of eNOS. The inhibition occurred very quickly (within 15min) and decreased the activity of Enos in a dose-dependent manner, resulting in the reduction of no synthesis, vasoconstriction and elevated blood pressure. The inhibition of eNOS activity by NaCl may be achieved by affecting Ca2 entry.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363
【参考文献】
相关期刊论文 前4条
1 刘景生，，强文安;一氧化氮合成酶及其调节[J];基础医学与临床;1996年06期
2 平庆功，严文魁;新型NO供体治疗心血管疾病和肺疾病[J];基础医学与临床;1996年06期
3 张东亚;内皮细胞松弛因子／－氧化氮病理生理及有关治疗[J];中国循环杂志;1996年02期
4 路萍 ,吕星 ,邢瑞云 ,周绪斌 ,郑晓飞 ,韩莉 ,汪毅荣 ,孙琪云;内皮细胞型一氧化氮合酶基因多态性与高血压病的相关性研究[J];中华心血管病杂志;2002年02期
本文编号：2165270
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2165270.html
最近更新
教材专著
