【摘要】：目的失重或模擬失重可導(dǎo)致心血管功能失調(diào),血管內(nèi)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(dǎo)血管內(nèi)皮功能異常中,氧化應(yīng)激水平增加有非常關(guān)鍵作用。本實(shí)驗(yàn)的目的是研究模擬失重條件下,應(yīng)用過氧化氫(H_2O_2)來刺激人臍靜脈內(nèi)皮細(xì)胞(EA.hy926),探討其對細(xì)胞凋亡的影響及其相關(guān)的機(jī)制,并對紅景天苷的細(xì)胞保護(hù)作用及機(jī)制進(jìn)行深入探討。方法EA.hy926細(xì)胞,用10%的胎牛血清的DMEM高糖培養(yǎng)液在5%CO2,37℃的細(xì)胞培養(yǎng)箱內(nèi)進(jìn)行細(xì)胞培養(yǎng),當(dāng)細(xì)胞傳代到四代后,隨機(jī)分為對照組(Con72)、回轉(zhuǎn)組(Z72),細(xì)胞回轉(zhuǎn)72h后,兩組細(xì)胞再隨機(jī)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(yù)(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(yù)(C+H_2O_2+Sal+LY和Z+H_2O_2+Sal+LY組)。以流式細(xì)胞儀器的方法檢測其細(xì)胞凋亡,采用二代測序技術(shù)對Con72、Con72+H_2O_2、Z72和Z72+H_2O_2四組細(xì)胞進(jìn)行轉(zhuǎn)錄測序,篩選凋亡相關(guān)差異基因,并以生物信息學(xué)技術(shù)進(jìn)行GO及KEGG分析,以RT-q PCR技術(shù)對篩選的差異基因進(jìn)行驗(yàn)證。在此基礎(chǔ)上,以RT-q PCR技術(shù)檢測紅景天苷作用下對差異基因表達(dá)的影響,分析其對抗模擬失重下H_2O_2誘導(dǎo)細(xì)胞凋亡的分子機(jī)制。結(jié)果通過流式細(xì)胞儀Annexin V-FITC/7-AAD法對細(xì)胞凋亡檢測結(jié)果表明模擬失重條件下經(jīng)過氧化氫刺激誘導(dǎo)EA.hy926細(xì)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(dǎo)的細(xì)胞凋亡,起到保護(hù)EA.hy926細(xì)胞的功效。轉(zhuǎn)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(yīng)用q RT-PCR技術(shù)驗(yàn)證發(fā)現(xiàn)BCL-2A1、FAM196B與轉(zhuǎn)錄組測序結(jié)果一致,其中BCL2A1為抗凋亡基因,FAM196B基因是促進(jìn)性因子,能夠促進(jìn)細(xì)胞增殖。同時發(fā)現(xiàn)紅景天苷干預(yù)后,BCL2A1、FAM196B基因顯著增加,說明紅景天苷可通過調(diào)控這2個基因發(fā)揮抗凋亡作用,保護(hù)模擬失重下過氧化氫誘導(dǎo)的內(nèi)皮細(xì)胞凋亡。結(jié)論模擬失重下EA.hy926細(xì)胞經(jīng)H_2O_2刺激后細(xì)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(dǎo)的細(xì)胞凋亡,其機(jī)制與紅景天苷調(diào)控BCL2A1及FAM196B基因表達(dá)相關(guān)。
[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(xué)位授予單位】：錦州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R85

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 孟京瑞，沈羨云，向求魯;兔模擬失重裝置的設(shè)計及其應(yīng)用[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;1996年01期

2 馮慶義,李俊峰,王天舒;水下模擬失重動力學(xué)研究綜述[J];力學(xué)與實(shí)踐;2004年03期

3 王俊鋒;郝叢軍;劉長庭;汪德生;袁明;;模擬失重時肺動脈反應(yīng)性變化的內(nèi)皮機(jī)制研究[J];解放軍醫(yī)學(xué)雜志;2007年05期

4 高放;張立藩;黃威權(quán);孫嵐;;慢性阻斷血管緊張素Ⅱ1型受體不能完全防止模擬失重大鼠血管的適應(yīng)性結(jié)構(gòu)變化[J];生理學(xué)報;2007年06期

5 章燁;袁明;李婷;張瑩;劉長庭;;模擬失重對肺微血管內(nèi)皮細(xì)胞凋亡的影響[J];軍醫(yī)進(jìn)修學(xué)院學(xué)報;2009年01期

6 何薇薇;王愛紅;賈化平;梁會澤;周環(huán)宇;邵將;魏相東;;頭低位臥床模擬失重狀態(tài)下房室平面運(yùn)動的定量組織速度成像[J];第四軍醫(yī)大學(xué)學(xué)報;2009年10期

7 周環(huán)宇;梁會澤;何薇薇;許永杰;魏相東;賈化平;王愛紅;;模擬失重對門靜脈血流動力學(xué)影響的彩色多普勒超聲研究[J];中華醫(yī)學(xué)超聲雜志(電子版);2010年05期

8 韓浩倫;吳瑋;薄少軍;丁瑞英;王鴻南;;豚鼠模擬失重的實(shí)驗(yàn)設(shè)計與應(yīng)用[J];總裝備部醫(yī)學(xué)學(xué)報;2011年02期

9 董麗;王瓊;劉新民;楊思進(jìn);;地面模擬失重實(shí)驗(yàn)方法概況[J];中國實(shí)驗(yàn)動物學(xué)報;2013年05期

10 王金華;模擬失重對人體作用的新方法[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;1989年01期

相關(guān)會議論文 前10條

1 孟芮;續(xù)惠云;狄升蒙;騫愛榮;商澎;;抗磁懸浮模擬失重對人骨髓間充質(zhì)干細(xì)胞的影響[A];中國空間科學(xué)學(xué)會第七次學(xué)術(shù)年會會議手冊及文集[C];2009年

2 郭云珠;尹大川;耿利強(qiáng);盧慧，

本文編號：2225524