SIRT1在低氧性肺血管平滑肌细胞中的表达及枸杞多糖的干预作用

发布时间：2018-04-18 05:17

本文选题：肺血管平滑肌细胞 + 低氧性肺动脉高压　；参考：《宁夏医科大学》2017年硕士论文

【摘要】：目的:观察在缺氧条件下进行枸杞多糖干预后肺血管平滑肌细胞中沉默接合型信息调节因子2同源蛋白1(SIRT1)、基质金属蛋白酶9(MMP-9)及缺氧诱导因子1α(HIF-1α)的表达。方法:肺血管平滑肌细胞分为常氧培养的正常细胞(200 m L/L氧气)、2 mg/m L枸杞多糖处理的常氧细胞、DMSO处理的常氧细胞、DMSO处理的缺氧细胞(100 m L/L氧气)、(0.5、1、2)mg/m L枸杞多糖处理的缺氧细胞、SIRT1的特异性激动剂白芦藜醇(10umol/L)处理的缺氧细胞、非特异性激动剂SR-1720(1umol/L)处理的缺氧细胞、SIRT1抑制剂EX-527(0.5umol/L)处理的缺氧细胞,培养于6、12、24 h,实时定量PCR、Western blot法分别检测SIRT1、MMP-9及HIF-1α的m RNA和蛋白表达量;在缺氧的肺血管平滑肌细胞中加入枸杞多糖处理12 h,实时定量PCR、Western blot法分别检测肺血管平滑肌细胞中SIRT1、MMP-9及HIF-1α的表达;最后按以上分组行划痕及Transwell小室实验。结果:1.缺氧条件下肺血管平滑肌细胞SIRT1 m RNA和蛋白水平降低,MMP-9及HIF-1αm RNA和蛋白水平升高。2.在缺氧条件下加入LBP可以提高SIRT1的表达,同时抑制MMP-9及HIF-1α的表达,且此作用具有浓度依赖性,随着低、中、高LBP浓度梯度的升高SIRT1的表达量逐渐增多,同时MMP-9、HIF-1α的表达量逐渐降低。3.SIRT1的特异性激动剂白藜芦醇可抑制MMP-9的表达。4.在缺氧条件下,肺血管平滑肌细胞的迁移及侵袭能力增强,而加入枸杞多糖后抑制了细胞的侵袭和迁移能力。结论:1.在缺氧条件下肺血管平滑肌细胞中SIRT1基因表达水平下调,而MMP-9及HIF-1α基因表达水平上调,我们推测在肺血管平滑肌细胞中SIRT1、MMP-9以及HIF-1α这三者表达量的变化与低氧性肺动脉高压的发生和发展具有一定的相关性。2.当在缺氧的肺血管平滑肌细胞中加入低、中、高剂量的LBP后,我们发现随着LBP浓度梯度的升高,SIRT1的表达量上升,而MMP-9及HIF-1α的表达反而随着LBP浓度梯度的升高而降低,这些告诉我们LBP可能对于低氧性肺动脉高压具有一定的改善作用,且它的这种作用主要是通过调节SIRT1及相关因子表达量的变化来实现的。3.SIRT1的激动剂白藜芦醇可以抑制MMP-9的表达,提示我们SIRT1、MMP-9及HIF-1α三者之间的表达具有一定的相关性。4.LBP可以抑制缺氧条件下血管平滑肌细胞的迁移和侵袭,这点告诉我们它可以抑制一些由于缺氧而引起的肺血管增殖以及重塑(如低氧性肺动脉高压)等疾病的发生。
[Abstract]:Aim: to observe the expression of silencing signal regulatory factor-2 homologous protein 1 sirt1, matrix metalloproteinase 9 (MMP-9) and hypoxia inducible factor-1 伪 (HIF-1 伪) in pulmonary vascular smooth muscle cells after treatment with Lycium barbarum polysaccharides (LBP) under hypoxia.Methods: pulmonary vascular smooth muscle cells were divided into normal cells cultured in normoxic oxygen (200ml 路L / L) and normal oxygen cells treated with 2 mg/m / L Lycium barbarum polysaccharides (LBP). DMSO-treated normoxic cells treated with DMSO-treated hypoxia cells (100ml / L) were treated with 100ml / L oxygen / L oxygen (0.5mg / mL) / mL Lycium barbarum polysaccharides (LBP).The anoxic cells were treated with 10 umol / L, a specific agonist of SIRT1.Hypoxia cells treated with SR-1720U 1umol/ L) and treated with SIRT1 inhibitor EX-5270.5umol/ L) were cultured for 24 h. The expression of m RNA and protein of SIRT1 MMP-9 and HIF-1 伪 were detected by real-time quantitative PCR Western blot.After treated with Lycium barbarum polysaccharides for 12 h, the expression of SIRT1MMP 9 and HIF-1 伪 in pulmonary vascular smooth muscle cells (VSMCs) were detected by real-time quantitative PCR Western blot assay. Finally, scratches and Transwell chamber experiments were performed according to the above groups.The result is 1: 1.Under hypoxia condition, the levels of SIRT1 m RNA and protein in pulmonary vascular smooth muscle cells decreased, and the levels of MMP-9 and HIF-1 伪 m RNA and protein increased.Under hypoxia condition, LBP could increase the expression of SIRT1 and inhibit the expression of MMP-9 and HIF-1 伪 in a concentration-dependent manner. With the increase of the gradient of LBP concentration, the expression of SIRT1 increased gradually.At the same time, the expression of MMP-9 and HIF-1 伪 decreased gradually. 3. Resveratrol, a specific agonist of SIRT1, inhibited the expression of MMP-9.Under hypoxia condition, the migration and invasion of pulmonary vascular smooth muscle cells were enhanced, but the ability of invasion and migration was inhibited by adding Lycium barbarum polysaccharides.Conclusion 1.The expression of SIRT1 gene was down-regulated in pulmonary vascular smooth muscle cells under hypoxia, while the expression of MMP-9 and HIF-1 伪 genes was up-regulated.We speculate that the changes of the expression of SIRT1 MMP-9 and HIF-1 伪 in pulmonary vascular smooth muscle cells may be related to the occurrence and development of hypoxic pulmonary hypertension.After adding low, medium and high doses of LBP into hypoxic pulmonary vascular smooth muscle cells, we found that the expression of SIRT1 increased with the increase of LBP concentration gradient, while the expression of MMP-9 and HIF-1 伪 decreased with the increase of LBP concentration gradient.These results suggest that LBP may have an improved effect on hypoxic pulmonary hypertension, and this effect is mainly achieved by regulating the expression of SIRT1 and related factors. 3. Resveratrol, an agonist of SIRT1, can inhibit the expression of MMP-9.4. LBP could inhibit the migration and invasion of vascular smooth muscle cells under hypoxia.This suggests that it inhibits pulmonary vascular proliferation and remodeling, such as hypoxic pulmonary hypertension, caused by hypoxia.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R544.1

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