紅景天苷對模擬失重下過氧化氫誘導EA.hy926細胞凋亡的抑制作用
發(fā)布時間：2018-09-06 06:24
【摘要】：目的失重或模擬失重可導致心血管功能失調,血管內皮功能異常在其中具有重要作用。研究證明,在模擬失重的條件下,誘導血管內皮功能異常中,氧化應激水平增加有非常關鍵作用。本實驗的目的是研究模擬失重條件下,應用過氧化氫(H_2O_2)來刺激人臍靜脈內皮細胞(EA.hy926),探討其對細胞凋亡的影響及其相關的機制,并對紅景天苷的細胞保護作用及機制進行深入探討。方法EA.hy926細胞,用10%的胎牛血清的DMEM高糖培養(yǎng)液在5%CO2,37℃的細胞培養(yǎng)箱內進行細胞培養(yǎng),當細胞傳代到四代后,隨機分為對照組(Con72)、回轉組(Z72),細胞回轉72h后,兩組細胞再隨機分組,同時分別給予以下處理:(1)不給予H_2O_2刺激(Con72和Z72組);(2)給予H_2O_2刺激5h(Con72+H_2O_2和Z72+H_2O_2組);(3)H_2O_2刺激5h的同時給予紅景天苷干預(C+H_2O_2+Sal和Z+H_2O_2+Sal組);(4)H_2O_2刺激5h的同時給予紅景天苷和PI3K通路抑制劑LY-294002干預(C+H_2O_2+Sal+LY和Z+H_2O_2+Sal+LY組)。以流式細胞儀器的方法檢測其細胞凋亡,采用二代測序技術對Con72、Con72+H_2O_2、Z72和Z72+H_2O_2四組細胞進行轉錄測序,篩選凋亡相關差異基因,并以生物信息學技術進行GO及KEGG分析,以RT-q PCR技術對篩選的差異基因進行驗證。在此基礎上,以RT-q PCR技術檢測紅景天苷作用下對差異基因表達的影響,分析其對抗模擬失重下H_2O_2誘導細胞凋亡的分子機制。結果通過流式細胞儀Annexin V-FITC/7-AAD法對細胞凋亡檢測結果表明模擬失重條件下經過氧化氫刺激誘導EA.hy926細胞能夠發(fā)生明顯凋亡,紅景天苷能夠抑制模擬失重條件下過氧化氫誘導的細胞凋亡,起到保護EA.hy926細胞的功效。轉錄組測序篩選出的6個顯著差異基因,分別為BCL-2A1、FAM196B、TMEM158、PPP1R16B、CXCL8、PPP1R3B。應用q RT-PCR技術驗證發(fā)現BCL-2A1、FAM196B與轉錄組測序結果一致,其中BCL2A1為抗凋亡基因,FAM196B基因是促進性因子,能夠促進細胞增殖。同時發(fā)現紅景天苷干預后,BCL2A1、FAM196B基因顯著增加,說明紅景天苷可通過調控這2個基因發(fā)揮抗凋亡作用,保護模擬失重下過氧化氫誘導的內皮細胞凋亡。結論模擬失重下EA.hy926細胞經H_2O_2刺激后細胞凋亡率顯著升高,中藥紅景天的有效活性成分紅景天苷能夠抑制模擬失重下H_2O_2刺激誘導的細胞凋亡,其機制與紅景天苷調控BCL2A1及FAM196B基因表達相關。
[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.
【學位授予單位】：錦州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R85
[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.
【學位授予單位】：錦州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R85
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