模拟微重力对斑马鱼胚胎发育及microRNA表达的影响
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摘要
太空环境是一个复杂、多变而极端的环境,具有多种辐射、微重力、弱磁场等特点,其中微重力是宇航员在太空环境中所面对的最大障碍之一,因此微重力的生物学效应的研究成为科学家们所关注的最重要的太空问题之一。
本论文以斑马鱼(Danio rerio)胚胎作为地面模拟微重力效应的研究模型,采用美国航天航空局(NASA)研制的微重力模拟系统(Rotary Cell Culture System, RCCS),对不同发育时期的斑马鱼胚胎进行模拟微重力处理,从表型和基因表达两个层面上探讨模拟微重力对斑马鱼产生的生物学效应。结果表明,在表型水平上,模拟微重力可以导致斑马鱼胚胎发育的畸形和死亡比率增加；体长在重力恢复的短期内变长,10天后恢复到正常水平；心率在微重力处理过程中及重力恢复的短期内有所加快,但7天后恢复到正常水平。在基因表达水平上,应用Inicroarray对斑马鱼胚胎在模拟微重力条件下的1nicroRNA表达谱进行分析。结果表明,在不同发育时期对斑马鱼胚胎进行模拟微重力处理,以及对发育时期相同的斑马鱼胚胎进行不同时长的模拟微重力处理,均可导致microRNA表达谱的改变；在三组不同的微重力处理实验中(8-72hpf,24-72hpf和24-48hpf),共发现9个microRNA的表达出现显著变化,用于后续1nicroRNA巴基因的预测和通路分析。靶预测结果表明,microRNA-204,-429和-22a表达上调,microRNA-16a表达下调,由于其靶基因sgcg与心肌细胞缺陷,肌肉萎缩,导致心律不齐,心脏功能下降有关,因此本研究发现的微重力引起的其对应的斑马鱼心率的增加,可能和这些microRNA上调有关。研究发现的microRNA-738,-133b,-9*和-133a表达上调,它们对应的靶基因Nrarpa,能间接影响notch通路和Wnt通路,而这两个通路都是机体形态发生和发育的重要机制,因此是否与模拟微重力产生的胚胎畸形率和死亡率增加有关联,是非常有必要进一步分析的问题。
另外,本论文还应用real-timePCR的技术,对前期模拟微重力蛋白表达谱研究中获得的差异表达蛋白质对应基因的表达水平进行了定量分析。被检测的基因包括肌肉型的肌酸激酶a (ckma),蛋白酶26s-非ATP酶8(psmd8),肌动蛋白2 (acta2),β2微管蛋白(tubb2c)以及抗增殖蛋白(prohibitin)。结果表明,上述基因在不同的模拟微重力处理后,肌动蛋白2 (acta2),β2微管蛋白(tubb2c)以及抗增殖蛋白(prohibitin)的1mRNA水平表达下调,与蛋白质表达趋势相同,肌肉型的肌酸激酶a (ckma),蛋白酶26s-非ATP酶8 (psmd8)的mRNA水平表达下调,与蛋白质表达趋势相反。推测在基因转录水平、转录后翻译水平上存在其它分子参与的调控机制,然而这种调控机制是否与microRNA调控相关尚有待于进一步研究。
Space conditions are a complex set of phenomena involving multi-radiation, microgravity and weak magnetic field etc. Amongst all these phenomena, microgravity is a constant physical factor astronauts must meet during space flight. Therefore, the mechanism of microgravity-induced biological effects is one of the most important issues in space biological studies.
In this paper, zebrafish (Danio rerio) embryos at different development stages were exposed to simulated microgravity, respectively, using a rotary cell culture system (RCCS) designed by National Aeronautics and Space Administration (NASA) of America. Biological effects of simulated microgravity on zebrafish embryos were investigated at the phenotypic and gene expression levels. Malformation rate and mortality rate were found increased after simulated microgravity exposure. Body length and heart rate were also increased during microgravity exposure and after a shot period of gravity recovery, but both returned to normal level after 10 days and 7 days of gravity recovery, respectively. At gene expression level, microRNA expression profiles of zebrafish embryos exposed to simulated-microgravity were analyzed using microarrays. Results demonstrated the microRNA expression profiles of zebrafish embryos varied, depending on the development stages of embyos exposed to simulated microgravity and the exposure time. All together, nine miRNAs showed significant changes after three different microgravity exposure (8-72hpf,24-72hpf and 24-48hpf), and were subsequently used in analysis of microRNA target and pathway prediction.
The results of target prediction shows that, microRNA-204,-429 and-22a expression up-regulated, microRNA-16a expression down-regulated, as to their target gene sgcg was related to cardiomyocyte defects, muscular atrophy, and leading to arrhythmia, decline in cardiac function, increasing of heart rate of zebrafish under microgravity in this paper might be related to these microRNAs up-regulated. microRNA-738,-133b,-9* and-133a expression up-regulated, their target gene Nrarpa indirectly effects notch pathway and Wnt pathway. These two pathways are the significant mechanism about morphogenesis and development, therefore, whether incr- easing of malformation rate and mortality rate was related to this significant mechanism needs further studies.
In addition, the mRNA expression levels of five differentially expressed proteins, obtained from our previous simulated-microgravity research, were analyzed using quantitative PCR (RT-qRCR). The five genes, inculding creatine kinase muscle a (ckmα), proteasome 26S subunit non-ATPase 8 (psmd8), actin alpha2 (αctα2), tubulin beta 2c(tubb2c) and prohibitin (prohibitin), displayed during different simulated-microgravity exposures, the mRNA expression levels of actin alpha2 (αctα2), tubulin beta 2c (tubb2c) and prohibitin (prohibitin) down-regulated, had the same trends to the proteins; the mRNA expression levels of creatine kinase muscle a (ckmα), proteasome 26S subunit non-ATPase 8 (psmd8) down-regulated, had different trends to the proteins. We supposed in the level of gene transcription and post-transcription translation existed regulated mechanism other molecular involved. However, whether this regulated mechanism was related to microRNA needs further studies.
本论文以斑马鱼(Danio rerio)胚胎作为地面模拟微重力效应的研究模型,采用美国航天航空局(NASA)研制的微重力模拟系统(Rotary Cell Culture System, RCCS),对不同发育时期的斑马鱼胚胎进行模拟微重力处理,从表型和基因表达两个层面上探讨模拟微重力对斑马鱼产生的生物学效应。结果表明,在表型水平上,模拟微重力可以导致斑马鱼胚胎发育的畸形和死亡比率增加；体长在重力恢复的短期内变长,10天后恢复到正常水平；心率在微重力处理过程中及重力恢复的短期内有所加快,但7天后恢复到正常水平。在基因表达水平上,应用Inicroarray对斑马鱼胚胎在模拟微重力条件下的1nicroRNA表达谱进行分析。结果表明,在不同发育时期对斑马鱼胚胎进行模拟微重力处理,以及对发育时期相同的斑马鱼胚胎进行不同时长的模拟微重力处理,均可导致microRNA表达谱的改变；在三组不同的微重力处理实验中(8-72hpf,24-72hpf和24-48hpf),共发现9个microRNA的表达出现显著变化,用于后续1nicroRNA巴基因的预测和通路分析。靶预测结果表明,microRNA-204,-429和-22a表达上调,microRNA-16a表达下调,由于其靶基因sgcg与心肌细胞缺陷,肌肉萎缩,导致心律不齐,心脏功能下降有关,因此本研究发现的微重力引起的其对应的斑马鱼心率的增加,可能和这些microRNA上调有关。研究发现的microRNA-738,-133b,-9*和-133a表达上调,它们对应的靶基因Nrarpa,能间接影响notch通路和Wnt通路,而这两个通路都是机体形态发生和发育的重要机制,因此是否与模拟微重力产生的胚胎畸形率和死亡率增加有关联,是非常有必要进一步分析的问题。
另外,本论文还应用real-timePCR的技术,对前期模拟微重力蛋白表达谱研究中获得的差异表达蛋白质对应基因的表达水平进行了定量分析。被检测的基因包括肌肉型的肌酸激酶a (ckma),蛋白酶26s-非ATP酶8(psmd8),肌动蛋白2 (acta2),β2微管蛋白(tubb2c)以及抗增殖蛋白(prohibitin)。结果表明,上述基因在不同的模拟微重力处理后,肌动蛋白2 (acta2),β2微管蛋白(tubb2c)以及抗增殖蛋白(prohibitin)的1mRNA水平表达下调,与蛋白质表达趋势相同,肌肉型的肌酸激酶a (ckma),蛋白酶26s-非ATP酶8 (psmd8)的mRNA水平表达下调,与蛋白质表达趋势相反。推测在基因转录水平、转录后翻译水平上存在其它分子参与的调控机制,然而这种调控机制是否与microRNA调控相关尚有待于进一步研究。
Space conditions are a complex set of phenomena involving multi-radiation, microgravity and weak magnetic field etc. Amongst all these phenomena, microgravity is a constant physical factor astronauts must meet during space flight. Therefore, the mechanism of microgravity-induced biological effects is one of the most important issues in space biological studies.
In this paper, zebrafish (Danio rerio) embryos at different development stages were exposed to simulated microgravity, respectively, using a rotary cell culture system (RCCS) designed by National Aeronautics and Space Administration (NASA) of America. Biological effects of simulated microgravity on zebrafish embryos were investigated at the phenotypic and gene expression levels. Malformation rate and mortality rate were found increased after simulated microgravity exposure. Body length and heart rate were also increased during microgravity exposure and after a shot period of gravity recovery, but both returned to normal level after 10 days and 7 days of gravity recovery, respectively. At gene expression level, microRNA expression profiles of zebrafish embryos exposed to simulated-microgravity were analyzed using microarrays. Results demonstrated the microRNA expression profiles of zebrafish embryos varied, depending on the development stages of embyos exposed to simulated microgravity and the exposure time. All together, nine miRNAs showed significant changes after three different microgravity exposure (8-72hpf,24-72hpf and 24-48hpf), and were subsequently used in analysis of microRNA target and pathway prediction.
The results of target prediction shows that, microRNA-204,-429 and-22a expression up-regulated, microRNA-16a expression down-regulated, as to their target gene sgcg was related to cardiomyocyte defects, muscular atrophy, and leading to arrhythmia, decline in cardiac function, increasing of heart rate of zebrafish under microgravity in this paper might be related to these microRNAs up-regulated. microRNA-738,-133b,-9* and-133a expression up-regulated, their target gene Nrarpa indirectly effects notch pathway and Wnt pathway. These two pathways are the significant mechanism about morphogenesis and development, therefore, whether incr- easing of malformation rate and mortality rate was related to this significant mechanism needs further studies.
In addition, the mRNA expression levels of five differentially expressed proteins, obtained from our previous simulated-microgravity research, were analyzed using quantitative PCR (RT-qRCR). The five genes, inculding creatine kinase muscle a (ckmα), proteasome 26S subunit non-ATPase 8 (psmd8), actin alpha2 (αctα2), tubulin beta 2c(tubb2c) and prohibitin (prohibitin), displayed during different simulated-microgravity exposures, the mRNA expression levels of actin alpha2 (αctα2), tubulin beta 2c (tubb2c) and prohibitin (prohibitin) down-regulated, had the same trends to the proteins; the mRNA expression levels of creatine kinase muscle a (ckmα), proteasome 26S subunit non-ATPase 8 (psmd8) down-regulated, had different trends to the proteins. We supposed in the level of gene transcription and post-transcription translation existed regulated mechanism other molecular involved. However, whether this regulated mechanism was related to microRNA needs further studies.
引文
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