本文關(guān)鍵詞：尿酸對血管內(nèi)皮細(xì)胞氧化應(yīng)激反應(yīng)的影響及其對細(xì)胞的損傷作用　出處：《中南大學(xué)》2012年博士論文　論文類型：學(xué)位論文

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【摘要】：目的 觀察尿酸(UA)對人血管內(nèi)皮細(xì)胞氧化應(yīng)激反應(yīng)、以及細(xì)胞凋亡與壞死的影響。方法 體外培養(yǎng)人臍靜脈血管內(nèi)皮細(xì)胞(HUVEC),以0、0.05、0.10、0.20、0.30g/L濃度UA分別作用6、12、24及48h,利用流式細(xì)胞儀檢測HUVEC細(xì)胞凋亡率、細(xì)胞壞死率、以及活性氧(ROS)含量；采用分光光度計檢測HUVEC的超氧陰離子(02-)含量；應(yīng)用ELISA法檢測HUVEC培養(yǎng)上清液中還原型酰胺腺嘌呤二核苷酸磷酸(NADPH)、黃嘌呤氧化酶(XO)、內(nèi)皮型一氧化氮合酶(eNOS)、環(huán)加氧酶-2(COX-2)、脂加氧酶(LOX)水平；同時檢測分別使用NADPH抑制劑DPI、XO抑制劑Allo、eNOS抑制劑eNOSR后上述指標(biāo)的變化。 結(jié)果 1.HUVEC與不同濃度UA(0、0.05、0.10、0.20、0.30g/L)共培養(yǎng)不同時間(6、12、24、48h)后,細(xì)胞凋亡率顯著性增加,且細(xì)胞凋亡率隨著UA濃度增加和作用時間延長而顯著上升；當(dāng)UA濃度為0.30g/L、以及作用時間為48h時,細(xì)胞壞死率顯著性增加。 2.HUVEC與不同濃度UA(0、0.05、0.10、0.20、0.30g／L)共培’養(yǎng)不同時間(6、12、24、48h)后,細(xì)胞內(nèi)ROS及02-表達(dá)顯著性增加,且隨著刺激濃度增加和作用時間延長而顯著上升；當(dāng)UA濃度為0.30g/L、以及作用時間為48h時,細(xì)胞內(nèi)ROS及02-表達(dá)明顯下降；ROS及02-表達(dá)量與HUVEC細(xì)胞凋亡率呈正相關(guān)關(guān)系。 3.HUVEC與不同濃度UA(0、0.05、0.10、0.20、0.30g/L)共培養(yǎng)24h后,NADPH、XO及eNOS表達(dá)量隨著UA濃度增加而顯著增加,其表達(dá)量與HUVEC細(xì)胞凋亡率高度正相關(guān)；而COX-2及LOX表達(dá)量無明顯變化,其表達(dá)量與HUVEC細(xì)胞凋亡率不相關(guān)。 4.UA與不同氧化酶抑制劑共刺激HUVEC24h后,UA可激活NADPH、XO、eNOS表達(dá),DPI抑制NADPH及eNOS表達(dá),Allo抑制XO及eNOS表達(dá),eNOSR抑制eNOS表達(dá),且DPI、Allo及eNOSR均可降低ROS及02-表達(dá)；UA+DPI+Allo組ROS及02-表達(dá)較UA+Allo組顯著性降低,但較UA+DPI組無顯著性差異；UA+DPI+eNOSR組ROS及02-表達(dá)較UA+DPI組及UA+eNOS組均顯著性降低。 結(jié)論 1.UA呈一定濃度依賴性和時間依賴性促進(jìn)HUVEC損傷。 2.UA主要通過活化氧化酶NADPH及eNOS從而激活氧化應(yīng)激反應(yīng)途徑導(dǎo)致HUVEC損傷。
[Abstract]:objective
To observe the effect of uric acid (UA) on oxidative stress in human vascular endothelial cells, and the effect of apoptosis and necrosis.
Human umbilical vein endothelial cells (HUVEC) cultured in vitro, with the concentration of 0,0.05,0.10,0.20,0.30g/L UA and 48h 6,12,24 respectively, using HUVEC flow cytometry to detect apoptosis rate, cell necrosis rate and reactive oxygen species (ROS) with superoxide anion content; Spectrophotometric Determination of HUVEC (02-) was detected by HUVEC ELISA; method in the supernatant reduced nicotinamide adenine dinucleotide phosphate (NADPH), xanthine oxidase (XO), endothelial nitric oxide synthase (eNOS), cyclooxygenase -2 (COX-2), Lipoxygenase (LOX) level; at the same time were detected by NADPH inhibitor DPI, XO inhibitor Allo, the changes of the above indicators the eNOS inhibitor eNOSR.
Result
1.HUVEC and UA of different concentrations (0,0.05,0.10,0.20,0.30g/L) were cultured for different time (6,12,24,48h), the apoptosis rate was significantly increased, and the apoptosis rate with the increase of UA concentration and the prolongation of time increased significantly; when the concentration of UA was 0.30g/L, and the reaction time was 48h, cell necrosis rate was significantly increased.
2.HUVEC with different concentrations of UA (0,0.05,0.10,0.20,0.30g / L) co culture "raised in different time (6,12,24,48h), intracellular expression of ROS and 02- were significantly increased, and with the stimulation of the concentration and time increased significantly; when the concentration of UA was 0.30g/L, and the reaction time was 48h, the expression of ROS and 02- cells decreased significantly ROS; and 02- expression and apoptosis rate of HUVEC cells was positively correlated.
3.HUVEC with different concentrations of UA (0,0.05,0.10,0.20,0.30g/L) co culture 24h, NADPH, XO and eNOS expression with UA concentration increased, and the expression of the apoptosis rate of HUVEC cells is highly correlated; but no significant change of COX-2 and LOX expression, and the expression of HUVEC cell apoptosis rate.
With different 4.UA oxidase inhibitors were stimulated HUVEC24h, UA can activate NADPH and XO, the expression of eNOS, DPI and eNOS Allo inhibited the expression of NADPH, inhibit the expression of XO and eNOS, eNOSR inhibits the expression of eNOS, and DPI, Allo and eNOSR can reduce the expression of 02- and ROS; ROS and 02- expression in UA+DPI+Allo group was significantly lower than that in group UA+Allo however, compared with the UA+DPI group had no significant difference between the expression of UA+DPI+eNOSR and 02-; ROS group compared with UA+DPI group and UA+eNOS group were significantly lower.
conclusion
1.UA has a certain concentration dependence and time dependence to promote HUVEC damage.
2.UA is mainly activated by activation oxidase NADPH and eNOS to activate the oxidative stress reaction pathway to cause HUVEC damage.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R363

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本文編號：1357999