斑马鱼p26s-8基因克隆及功能研究
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摘要
太空环境中微重力条件会导致基因和蛋白表达谱发生变化。前期蛋白双向电泳结果初步表明,模拟微重力处理会引起斑马鱼P26S-8蛋白表达发生显著变化,Q-PCR结果表明p26s-8基因表达发生显著下调。因此,推测该基因是微重力感受基因。目前,关于p26s-8基因的功能研究较少,在斑马鱼中该基因尚未被克隆及研究。
本文以斑马鱼(Danio rerio)为模式生物,利用NASA研制的微重力模拟系统RCCS(Rotary Cell Culture System, RCCS)进行地面模拟。从p26s-8基因全序列的克隆、对应蛋白在微重力条件下表达变化的定量分析及其在细胞周期调控中的作用三方面,对微重力感受基因p26s-8进行了深入的研究。
本论文首次克隆获得了斑马鱼p26s-8基因的全序列,并对序列进行了初步分析及构建了进化树。对P26S-8蛋白在模拟微重力处理前后表达水平的定量分析表明：模拟微重力处理24hpf-48hpf和24hpf-72hpf的斑马鱼胚胎P26S-8蛋白水平发生显著上调,分别为处理组的1.18和1.22倍。同时发现,模拟微重力处理会导致斑马鱼胚胎的孵化产生延迟。利用斑马鱼胚胎成纤维细胞系ZF4进行细胞周期检测的结果表明：模拟微重力处理会使ZF4细胞的细胞周期阻断在G1期,抑制P26S-8蛋白也会导致细胞周期阻滞在G1。模拟微重力处理是否通过影响p26s-8对斑马鱼细胞G1-S期的调控,进而导致胚胎发育的延迟,尚待进一步验证。本课题获得的研究结果,为进一步揭示微重力产生的生物学效应机制提供了数据。
Microgravity environment of space can induce a serial of changes in gene and protein expression profilings. Our previous study of simulated-microgravity on zebrafish (Danio rerio) embryos demonstrated P26S-8protein was up-regulated on two-dimensional (2-D) electrophoresis gel, while the gene expression level of p26s-8was significantly decreased after simulated-microgravity. These results suggest that p26s-8may be a microgravity sensitive gene. However, functional study on p26s-8is very limited and it has not been cloned in zebrafish till now.
In this study, we tried to clone p26s-8gene in zebrafish, quantify its protein expression level in zebrafish embryos after simulated microgravity, and identify its function in cell cycle regulation. A rotary cell culture system (RCCS) designed by national aeronautics and apace administration (NASA) of America was used to simulate microgravity.
The whole sequence of p26s-8gene in zebrafish was cloned in this study for the first time. Preliminary analysis on its sequence and phylogenetic tree construction were carried out subsequently. Quantitative analysis by western blot showed that p26s-8protein expression levels were significantly increased1.18and1.22times after24-48hpf and24-72hpf simulated microgravity, respectively. Moreover, a significant delay on zebrafish embryo development was found in simulated-microgravity exposed group. Inhibition of P26S-8protein in zebrafish embryonic cell lines ZF4could block cell cycle in G1phase, which indicated that p26s-8may play a role in cell cycle regulation. Intererstingly, simulated-microgravity could also lead to the block in the G1. Whether it is p26s-8mediated cell cycle regulation result in the zebrafish embryo development delay after simulated microgravity exposure still needs further study. However, results obtained in this study will contribute to the biological mechanism revealing of microgravity.
本文以斑马鱼(Danio rerio)为模式生物,利用NASA研制的微重力模拟系统RCCS(Rotary Cell Culture System, RCCS)进行地面模拟。从p26s-8基因全序列的克隆、对应蛋白在微重力条件下表达变化的定量分析及其在细胞周期调控中的作用三方面,对微重力感受基因p26s-8进行了深入的研究。
本论文首次克隆获得了斑马鱼p26s-8基因的全序列,并对序列进行了初步分析及构建了进化树。对P26S-8蛋白在模拟微重力处理前后表达水平的定量分析表明：模拟微重力处理24hpf-48hpf和24hpf-72hpf的斑马鱼胚胎P26S-8蛋白水平发生显著上调,分别为处理组的1.18和1.22倍。同时发现,模拟微重力处理会导致斑马鱼胚胎的孵化产生延迟。利用斑马鱼胚胎成纤维细胞系ZF4进行细胞周期检测的结果表明：模拟微重力处理会使ZF4细胞的细胞周期阻断在G1期,抑制P26S-8蛋白也会导致细胞周期阻滞在G1。模拟微重力处理是否通过影响p26s-8对斑马鱼细胞G1-S期的调控,进而导致胚胎发育的延迟,尚待进一步验证。本课题获得的研究结果,为进一步揭示微重力产生的生物学效应机制提供了数据。
Microgravity environment of space can induce a serial of changes in gene and protein expression profilings. Our previous study of simulated-microgravity on zebrafish (Danio rerio) embryos demonstrated P26S-8protein was up-regulated on two-dimensional (2-D) electrophoresis gel, while the gene expression level of p26s-8was significantly decreased after simulated-microgravity. These results suggest that p26s-8may be a microgravity sensitive gene. However, functional study on p26s-8is very limited and it has not been cloned in zebrafish till now.
In this study, we tried to clone p26s-8gene in zebrafish, quantify its protein expression level in zebrafish embryos after simulated microgravity, and identify its function in cell cycle regulation. A rotary cell culture system (RCCS) designed by national aeronautics and apace administration (NASA) of America was used to simulate microgravity.
The whole sequence of p26s-8gene in zebrafish was cloned in this study for the first time. Preliminary analysis on its sequence and phylogenetic tree construction were carried out subsequently. Quantitative analysis by western blot showed that p26s-8protein expression levels were significantly increased1.18and1.22times after24-48hpf and24-72hpf simulated microgravity, respectively. Moreover, a significant delay on zebrafish embryo development was found in simulated-microgravity exposed group. Inhibition of P26S-8protein in zebrafish embryonic cell lines ZF4could block cell cycle in G1phase, which indicated that p26s-8may play a role in cell cycle regulation. Intererstingly, simulated-microgravity could also lead to the block in the G1. Whether it is p26s-8mediated cell cycle regulation result in the zebrafish embryo development delay after simulated microgravity exposure still needs further study. However, results obtained in this study will contribute to the biological mechanism revealing of microgravity.
引文
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[3]Synthecon incorporated. The Rotary Cell Culture SystemTM-The Premier Tissue Engineering Culture System. User's Guide.
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[13]冯岱雅.失重对人体心血管系统的影响及仿真.2005,25(3)：253-256
[14]马进,张立藩,杨天德.模拟失重大鼠动脉平滑肌收缩反应性的变化.中华航空航天医学杂志.1999(2)：37.
[15]孙磊,向求鲁,汪德生等.微重力与模拟重力条件下的肺循环变化.航天医学与医学工程. 2000,1 3(04)：305-309.
[16]孙联文.微重力导致航天员骨质疏松的研究进展.中华航空航天医学杂志.2004,5(1)：54-62.
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[18]孙怡宁.微重力引起骨丢失的细胞机制.国际骨科学杂志.2006,27(2)：126-128.
[19]Clement G. Musculo-skeletal system in space. Fundamentals of space medicine. 2003,173-204.
[20]Bateman TA. Countryman S1 Osteoprote gerin and bone loss associated with spaceflightl DDT.2002,7(8):456-457.
[20]Robert H. Fitts. Microgravity and skeletal muscle. J Appl Physiol.2000, 89:823-839.
[21]DANNY A. RILEY. Decreased thin filament density and length in human atrophic soleus muscle fibers after spaceflight. J. Appl. Physiol.2000,88:567-572.
[22]G. R. Adams. Effects of spaceflight and thyroid deficiency on hindlimb development I. Muscle mass and IGF-I expression. Appl. Physiol.2000,88:894-903.
[23]Buravkova LB, Rykova MP, Grigorieva V. Cell interactions in microgravity: cytotoxic effects of natural killer cells in vitro, J Gravit Physiol,2004,11(2): 177-180.
[24]Taylor W E, Bhasin S, Lalani R. Alteration of gene expression profiles in skeletal muscle of rats exposed to microgravity during a spaceflight. J Gravit Physiol.2002, 9:61-70.
[25]Tobin BW, Uchakin PN, Leeper-Woodford SK. Insuilin secretion and sensitivity in space light:diabetogenic effects. Nutrition.2002,18:842-848.
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