Gene expression profiling of human epidermal keratinocytes in simulated microgravity and recovery cultures

Simulated microgravity （SMG） bioreactors and DNA microarray technology are powerful tools to identify ＂space genes＂ that play key roles in cellular response to microgravity. We applied these biotechnology tools to investigate SMG and post-SMG recovery effects on human epidermal keratinocytes by exposing cells to SMG for 3, 4, 9, and 10 d using the high aspect ratio vessel bioreactor followed by recovery culturing for 15, 50, and 60 d in normal gravity. As a result, we identified 162 differentially expressed genes, 32 of which were ＂center genes＂ that were most consistently affected in the time course experiments. Eleven of the center genes were from the integrated stress response pathways and were coordinately downregulated. Another seven of the center genes, which are all metallothionein MT-Ⅰ and MT-Ⅱ isoforms, were coordinately up-regulated. In addition, HLA-G, a key gene in cellular immune response suppression, was found to be significantly upregulated during the recovery phase. Overall, more than 80% of the differentially expressed genes from the shorter exposures （≤4 d） recovered in 15 d; for longer （≥9 d） exposures, more than 50 d were needed to recover to the impact level of shorter exposures. The data indicated that shorter SMG exposure duration would lead to quicker and more complete recovery from the microgravity effect.     

Effects of clinorotation on COL1A1- EGFP gene expression

Itisnowwellestablishedthatspaceflightin-ducesbonelossafterlong-termexposuretomicro-gravity.Biochemicalstudiesshowedthatcontinuousboneloss[1,2]andmineralredistribution[3]inducedbyspaceflightwereassociatedwiththedecreasedos-teoblasticactivityanddifferentialfunction[4—6].Recentfindingssuggestthechangeintheexpressionofbone-formationrelatedgeneplaysanimportantroleintheprocessofdecreasedosteoblasticfunctionsin-ducedbyspaceflight.CollagentypeIexpressedthroughouttheprocessofosteoblasticproliferationa…     

Effect of culture in a rotating wall bioreactor on the physiology of differentiated neuron-like PC12 and SH-SY5Y cells.

A variety of evidence suggests that nervous system function is altered during microgravity, however, assessing changes in neuronal physiology during space flight is a non-trivial task. We have used a rotating wall bioreactor with a high aspect ratio vessel (HARV), which simulates the microgravity environment, to investigate the how the viability, neurite extension, and signaling of differentiated neuron-like cells changes in different culture environments. We show that culture of differentiated PC12 and SH-SY5Y cells in the simulated microgravity HARV bioreactor resulted in high cell viability, moderate neurite extension, and cell aggregation accompanied by NO production. Neurite extension was less than that seen in static cultures, suggesting that less than optimal differentiation occurs in simulated microgravity relative to normal gravity. Cells grown in a mixed vessel under normal gravity (a spinner flask) had low viability, low neurite extension, and high glutamate release. This work demonstrates the feasibility of using a rotating wall bioreactor to explore the effects of simulated microgravity on differentiation and physiology of neuron-like cells.     

间断性人工重力对模拟失重大鼠颈总动脉平滑肌细胞凋亡的影响

目的:研究3周模拟失重大鼠颈总动脉平滑肌细胞凋亡的变化及间断性人工重力对其的影响。方法:以尾部悬吊大鼠(SUS)模拟失重,同期每天悬吊23h、站立1h(STD)模拟间断性人工重力的对抗效果,用M30染色及Tunel染色方法观察3周SUS组、同步对照(CON)组及STD组颈总动脉平滑肌细胞早期和中晚期的凋亡情况,并用免疫组织化学方法及Western blot印迹方法观察各组大鼠颈总动脉组织Caspase-3的蛋白表达变化。结果:与CON组比较,SUS组大鼠颈总动脉平滑肌细胞M30染色阳性细胞明显减少,STD组M30染色阳性细胞较CON组及SUS组显著增加;SUS组Tunel染色阳性细胞较CON组及STD组显著减少,STD组Tunel染色阳性细胞较CON组及SUS组显著增加;SUS组Caspase-3的表达较CON组显著降低(P<0.05),STD组Caspase-3的表达较CON组及SUS组显著增高(P<0.01)。结论:模拟失重可引起大鼠颈总动脉平滑肌细胞凋亡减少,每日1 h的-Gx对抗使颈总动脉的凋亡增加。Caspase-3可能在调控模拟失重所致血管组织平滑肌细胞的凋亡中发挥作用。     

模拟微重力条件下 WB-F344细胞的三维培养

与传统的单层平面培养相比,细胞三维培养可更好地模拟生物体内细胞的生长状态和微环境.以Cytodex-3微载体为支持物,利用旋转式细胞培养系统（RCCS）模拟微重力条件,悬浮培养法构建大鼠WB-F344细胞微重力三维培养模型.并通过细胞计数、光学显微镜、透射电镜、逆转录-聚合酶链反应（RT-PCR）和流式细胞术等方法分析了细胞增殖、显微结构、粘附分子及钙粘蛋白（E-cadherin）表达情况.结果表明,模拟微重力三维培养条件下WB-F344细胞增殖块,呈紧密多层排列、可见丰富的微绒毛和线粒体、胞间有桥粒和紧密连接形成,细胞粘着力加强、表现出良好的三维生长特征;与静置三维培养相比,纤粘连蛋白（Fn）mRNA表达呈上调趋势,细胞内E-cadherin表达量增加,这可能是微重力效应下细胞粘附力增强的部分机制.该培养体系可能有利于细胞之间,细胞与胞外基质之间相互作用及其作用机制的研究.     

Growth and Development in Arabidopsis thaliana through an Entire Life Cycle under Simulated Microgravity Conditions on a Clinostat

Arabidopsis thaliana ecotype Columbia and Landsberg erecta were studied. Horizontal clinorotation affected little germination of seeds, growth and development of rosette leaves and roots during early vegetative growth stage, and the onset of the bolting of inflorescence axis and flower formation in reproductive growth stage, although it suppressed elongation of inflorescence axes. The clinorotation substantially reduced the numbers of siliques and seeds in Landsberg erecta, and completely inhibited seed production in Columbia. Seeds produced in Landsberg erecta on the clinostat were capable of germinating and developing rosette leaves normally on the ground. On the other hand, growth of pin-formed mutant (pin/pin) of Arabidopsis ecotype Enkheim, which has a unique structure of inflorescence axis with no flower and extremely low levels of auxin polar transport activity, was inhibited and the seedlings frequently died during vegetative stage on the clinostat. Seed formation and inflorescence growth of the seedlings with normal shape (pin/+ or +/+) were also suppressed on the clinostat. These results suggest that the growth and development of Arabidopsis, especially in reproductive growth stage, is suppressed under simulated microgravity conditions on a clinostat. To complete the life cycle probably seems to be quite difficult, although it is possible in some ecotypes. Received 18 June 1999/ Accepted in revised form 27 August 1999     

Effect of simulated microgravity induced PI3K-nos2b signalling on zebrafish cardiovascular plexus network formation

Local abnormal angiogenesis and cardiovascular system reorganization have been observed in embryos exposed to a simulated microgravity (SM) environment. In this study, changes in key molecular signals and pathways in cardiovascular development have been investigated under microgravity conditions. In particular, the caudal vein plexus (CVP) network, formed by sprouting angiogenesis has been chosen. Zebrafish embryos were exposed to SM using a ground-based microgravity bioreactor for 24 and 36 h. The SM was observed to have no effect on the zebrafish length, tail width and incubation time whereas it was observed to significantly reduce the heart rate frequency and to promote abnormal development of the CVP network in the embryos. Nitric oxide (NO) content demonstrated that the total proteins in zebrafish embryos were significantly higher in SM than in the control group grown under normal conditions. It was then preliminarily determined how NO signals were involved in SM regulated zebrafish CVP network formation. nos2b MO was injected and CVP network evolution was observed in 36 h post fertilization (hpf) under SM condition. The results showed that the CVP network formation was considerably decreased in the nos2b MO treated group. However, this inhibition of the CVP network development was not observed in control MO group, indicating that nos2b is involved in the SM-regulated vascular development process in zebrafish. Moreover, specific phosphoinositide 3-kinase (PI3K) inhibitors such as LY294002 were also tested on zebrafish embryos under SM condition. This treatment significantly inhibited the formation of zebrafish CVP network. Furthermore, overexpression of nos2b partly rescued the LY294002-caused CVP network failure. Therefore, it can be concluded that SM affects zebrafish CVP network remodeling by enhancing angiogenesis. Additionally, the PI3K-nos2b signaling pathway is involved in this process.     

Effects of microgravity on the larval development, metamorphosis and reproduction of the urodele amphibian Pleurodeles waltl

The FERTILE experiment was twice performed onboard the Mir space station during the Cassiopée and Pégase French space missions. The goal was to analyze the effects of microgravity on fertilization and embryonic development, and then on further development on the ground in the amphibian Pleurodeles waltl. The present paper reports development that occurred in the laboratory after landing. Recovered on the ground at the hatching stage, young larvae reared at room temperature underwent metamorphosis and became adults without obvious abnormalities. Of particular interest was the rearing temperature that induced a delayed metamorphosis for animals from the Cassiopée space mission, but not for animals from the Pégase mission. The rate of development and the morphology were analogous in these animals and in ground controls reared in a similar annual period. Analysis of offspring was performed using these animals. Males born in space were first mated with control ground-born females and then with females born in space. The mating gave progeny that developed normally. Depending on the methods used and on the limits of the analyses, the results clearly demonstrated that animals born in space were able to live and reproduce after return to the ground.     

模拟微重力环境因子对人参细胞生长和人参皂苷含量的影响

与在正常重力条件培养下的对照相比,经回转器水平回转处理的人参细胞鲜重和干重均增加,人参皂苷含量提高10％左右。在去Ca2 培养基上生长的人参愈伤组织细胞,经回转器水平回转3周后,人参皂苷含量约为正常重力条件下培养细胞的2倍。另外,在试验范围内,如果培养基中起始钙离子浓度越高,则其培养的人参细胞中人参皂苷含量越低。