氯化鈉抑制內(nèi)皮型一氧化氮合成酶活性及其機(jī)制探討
發(fā)布時間：2018-08-04 21:49
【摘要】： 目的 通過內(nèi)皮型一氧化氮合成酶(endothelial nitric oxide synthase, eNOS)活性分析證明:微量氯化鈉(sodium chloride, NaCl)濃度(5～20mmol/L)的升高能夠顯著抑制eNOS活性,使一氧化氮(nitric oxide, NO)合成減少。通過乙酰膽堿(acetylcholine, Ach)和鈣離子載體A23187(eNOS激活劑)做對照,進(jìn)一步探討NaCl抑制eNOS活性的分子機(jī)制。 方法 1.細(xì)胞實驗:應(yīng)用原代小牛動脈內(nèi)皮細(xì)胞(primary bovine endothelial cells, BA EC)及表達(dá)eNOS或者vector的中國倉鼠卵巢細(xì)胞(Chinese hamster ovary cells, CHO)進(jìn)行Western Blot及eNOS活性分析。 2.排除實驗干擾因素:應(yīng)用等毫摩爾甘露醇代替NaCl培養(yǎng)的BAEC細(xì)胞系進(jìn)行eNOS活性分析、應(yīng)用BAEC細(xì)胞系進(jìn)行WST染色及3H-L-精氨酸攝取能力實驗,排除滲透壓、細(xì)胞活性改變及3H-L-精氨酸攝取能力改變對實驗結(jié)果的影響。 3.動脈內(nèi)皮細(xì)胞再生分析:應(yīng)用eNOS抑制劑NG-硝基-L-精氨酸甲酯(NG-Nitro- L- arginine Methyl Ester, L-NAME)和NaCl分別培養(yǎng)小鼠主動脈環(huán),觀察NaCl對小鼠動脈內(nèi)皮細(xì)胞再生的影響。 4.延伸實驗:分別用Ach、Ach加NaCl、A23187、A23187加NaCl培養(yǎng)的細(xì)胞分析eNOS活性,進(jìn)一步探討NaCl抑制eNOS活性的分子機(jī)制。 結(jié)果 1.通過BAEC、CHO-eNOS、CHO-vector細(xì)胞系進(jìn)行Western Blot顯示:CHO-vector細(xì)胞系不表達(dá)eNOS蛋白,而BAEC和CHO-eNOS細(xì)胞系表達(dá)eNOS蛋白,說明可以用后兩種細(xì)胞系進(jìn)行eNOS活性分析。 2.通過應(yīng)用表達(dá)eNOS蛋白的BAEC細(xì)胞系和CHO-eNOS細(xì)胞系進(jìn)行eNOS活性分析發(fā)現(xiàn):培養(yǎng)細(xì)胞所用的NaCl濃度越高,eNOS活性越低(P0.0001),NaCl對eNOS活性的抑制呈劑量依賴關(guān)系。實驗中,NaCl孵育細(xì)胞15min就能顯著抑制eNOS活性。 3.通過應(yīng)用等毫摩爾甘露醇代替不同濃度NaCl培養(yǎng)細(xì)胞進(jìn)行eNOS活性分析,證明eNOS活性的抑制不是由于加入NaCl后滲透壓改變引起的(P0.05);通過對加NaCl(137～157mmol/L)培養(yǎng)的BAEC細(xì)胞系進(jìn)行WST染色,發(fā)現(xiàn)NaCl培養(yǎng)組細(xì)胞存活率與PBS培養(yǎng)組相比無顯著性差異(P0.05);通過應(yīng)用BAEC細(xì)胞系進(jìn)行3H-L-精氨酸攝取能力分析(3H-L-Arginine uptake assay),證明不同濃度NaCl對L-精氨酸進(jìn)入細(xì)胞的量無顯著性影響(P0.05)。 4.通過進(jìn)行動脈內(nèi)皮細(xì)胞再生實驗,發(fā)現(xiàn)培養(yǎng)液中加入eNOS抑制劑L-NAME后,動脈內(nèi)皮細(xì)胞生長非常緩慢。培養(yǎng)液中加入NaCl的二組細(xì)胞同樣生長緩慢,尤其是加入的NaCl濃度為20mmol/L的一組,內(nèi)皮細(xì)胞生長狀況近似于L-NAME組。 5.NaCl抑制eNOS活性的分子機(jī)制的研究:發(fā)現(xiàn)eNOS激活劑Ach和A23187培養(yǎng)的細(xì)胞eNOS活性增強(qiáng),但是同時加入NaCl培養(yǎng)細(xì)胞后,增強(qiáng)的eNOS活性被抑制,并且NaCl濃度越高,對eNOS活性的抑制越顯著(P0.05)。 結(jié)論 1.微量NaCl濃度的升高(5～20mmol/L)就能顯著抑制eNOS活性,這種抑制發(fā)生的非常迅速(15min內(nèi))并且呈劑量依賴關(guān)系。eNOS活性降低使NO合成減少,血管收縮,血壓升高。 2. NaCl對eNOS活性的抑制可能是通過影響Ca2+入胞實現(xiàn)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.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R363
本文編號：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.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R363
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