红景天苷对模拟失重下过氧化氢诱导EA.hy926细胞凋亡的抑制作用

发布时间：2018-09-06 06:24

【摘要】：目的失重或模拟失重可导致心血管功能失调,血管内皮功能异常在其中具有重要作用。研究证明,在模拟失重的条件下,诱导血管内皮功能异常中,氧化应激水平增加有非常关键作用。本实验的目的是研究模拟失重条件下,应用过氧化氢(H_2O_2)来刺激人脐静脉内皮细胞(EA.hy926),探讨其对细胞凋亡的影响及其相关的机制,并对红景天苷的细胞保护作用及机制进行深入探讨。方法EA.hy926细胞,用10%的胎牛血清的DMEM高糖培养液在5%CO2,37℃的细胞培养箱内进行细胞培养,当细胞传代到四代后,随机分为对照组(Con72)、回转组(Z72),细胞回转72h后,两组细胞再随机分组,同时分别给予以下处理:(1)不给予H_2O_2刺激(Con72和Z72组);(2)给予H_2O_2刺激5h(Con72+H_2O_2和Z72+H_2O_2组);(3)H_2O_2刺激5h的同时给予红景天苷干预(C+H_2O_2+Sal和Z+H_2O_2+Sal组);(4)H_2O_2刺激5h的同时给予红景天苷和PI3K通路抑制剂LY-294002干预(C+H_2O_2+Sal+LY和Z+H_2O_2+Sal+LY组)。以流式细胞仪器的方法检测其细胞凋亡,采用二代测序技术对Con72、Con72+H_2O_2、Z72和Z72+H_2O_2四组细胞进行转录测序,筛选凋亡相关差异基因,并以生物信息学技术进行GO及KEGG分析,以RT-q PCR技术对筛选的差异基因进行验证。在此基础上,以RT-q PCR技术检测红景天苷作用下对差异基因表达的影响,分析其对抗模拟失重下H_2O_2诱导细胞凋亡的分子机制。结果通过流式细胞仪Annexin V-FITC/7-AAD法对细胞凋亡检测结果表明模拟失重条件下经过氧化氢刺激诱导EA.hy926细胞能够发生明显凋亡,红景天苷能够抑制模拟失重条件下过氧化氢诱导的细胞凋亡,起到保护EA.hy926细胞的功效。转录组测序筛选出的6个显著差异基因,分别为BCL-2A1、FAM196B、TMEM158、PPP1R16B、CXCL8、PPP1R3B。应用q RT-PCR技术验证发现BCL-2A1、FAM196B与转录组测序结果一致,其中BCL2A1为抗凋亡基因,FAM196B基因是促进性因子,能够促进细胞增殖。同时发现红景天苷干预后,BCL2A1、FAM196B基因显著增加,说明红景天苷可通过调控这2个基因发挥抗凋亡作用,保护模拟失重下过氧化氢诱导的内皮细胞凋亡。结论模拟失重下EA.hy926细胞经H_2O_2刺激后细胞凋亡率显著升高,中药红景天的有效活性成分红景天苷能够抑制模拟失重下H_2O_2刺激诱导的细胞凋亡,其机制与红景天苷调控BCL2A1及FAM196B基因表达相关。
[Abstract]:Objective Weightlessness or simulated weightlessness can lead to cardiovascular dysfunction, in which vascular endothelial dysfunction plays an important role. Studies have shown that under simulated weightlessness, increased levels of oxidative stress play a very important role in inducing vascular endothelial dysfunction. H_2O_2 stimulated human umbilical vein endothelial cells (EA.hy926) to explore the effect of H_2O_2 on apoptosis and its related mechanism, and to explore the cytoprotective effect and mechanism of salidroside. Methods EA.hy926 cells were cultured in DMEM high glucose medium with 10% fetal bovine serum at 5% CO_2 and 37 C, and the cells were fine. After passage to the fourth generation, the cells were randomly divided into control group (Con72) and rotary group (Z72). After 72 hours of cell rotation, the cells of the two groups were randomly divided into the following groups: (1) no H_2O_2 stimulation (Con72 and Z72 groups); (2) H_2O_2 stimulation for 5 hours (Con72+H_2O_2 and Z72+H_2O_2 groups); (3) H_2O_2 stimulation for 5 hours and Salidroside intervention (C+H_2O_2+Z72+H_2O_2+H_2) Sal and Z+H_2O_2+Sal groups; (4) Salidroside and PI3K pathway inhibitor LY-294002 were administered at the same time of stimulation with H_2O_2 for 5 hours (C+H_2O_2+Sal+LY and Z+H_2O_2+Sal+LY groups). Cell apoptosis was detected by flow cytometry. Con72, Con72+H_2O_2, Z72 and Z72+H_2O_2 groups were transcribed and sequenced by second-generation sequencing technique. The differentially expressed genes related to death were analyzed by GO and KEGG with bioinformatics techniques, and the differentially screened genes were verified by RT-q PCR. On this basis, the effects of salidroside on the expression of differentially expressed genes were detected by RT-q PCR, and the molecular mechanism of its resistance to H_2O_2-induced apoptosis under simulated weightlessness was analyzed. The results of flow cytometry Annexin V-FITC/7-AAD showed that EA.hy926 cells could be induced to apoptosis by hydrogen oxide stimulation under simulated weightlessness. Salidroside could inhibit the apoptosis induced by hydrogen peroxide under simulated weightlessness and protect EA.hy926 cells. Transcription group sequencing screened out The results of QRT-PCR analysis showed that BCL-2A1 and FAM196B were identical with transcriptome sequencing. BCL-2A1 was an anti-apoptotic gene, FAM196B was a promoter and could promote cell proliferation. The results showed that salidroside could protect endothelial cells from apoptosis induced by hydrogen peroxide under simulated weightlessness by regulating these two genes. Conclusion The apoptosis rate of EA.hy926 cells stimulated by H_2O_2 increased significantly under simulated weightlessness. Salidroside, an active component of Rhodiola, could inhibit simulated weightlessness. The mechanism of H_2O_2-induced apoptosis is related to the regulation of BCL2A1 and FAM196B gene expression by salidroside.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R85

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