模拟微重力对人骨髓间充质干胞向成骨细胞分化中细胞信号通路影响的分析

通过模拟来研究微重力对hMSC向成骨细胞分化的影响,并利用相关信号通路的激活剂或抑制剂来调节这一分化过程．研究结果表明,在成骨细胞分化诱导条件下,微重力降低了hMSC向成骨细胞定向分化的能力,并且成骨细胞标记性基因的表达明显降低,Runt相关转录因子2（Runx2）的表达受到抑制．相反,过氧化物酶体增殖激活受体γ（PPARγ2）的表达则增加．同时,微重力也降低了ERK的磷酸化水平,而增加了p38MAPK的磷酸化水平．使用p38MAPK的抑制剂SB203580能够抑制p38MAPK的磷酸化,但不能降低PPARγ2的表达水平．骨形态发生蛋白（BMP）能增加Runx2的表达水平．成纤维细胞生长因子2（FGF2）增加了ERK的磷酸化水平,但也不能显著增加成骨细胞标记性基因的表达水平．采用BMP,FGF2和SB203580三种因子组合来调控微重力下的成骨细胞分化能力,结果表明三者的协同作用能显著逆转微重力对成骨细胞定向分化的生物学效应．研究结果还说明,模拟微重力应该是通过不同的细胞信号通路来抑制成骨细胞分化和提升脂肪细胞分化的．     

植物细胞响应重力变化的Ca2+信号

重力大小的改变(微重力和超重力)可以对植物的生长发育、生理生化特性、细胞超微结构、基因和蛋白的表达等产生广泛的影响,而Ca~(2+)可能在此过程中起信号物质的作用.重力刺激在植物细胞中引发事件的顺序可能是:重力刺激的感受—细胞膜系统张力改变—膜理化特性改变—膜透性、离子转运、膜连接酶活性等改变—Ca~(2+)信号的产生和转导—新陈代谢变化—生理反应.植物对重力水平变化应激响应的至关重要一步是引起细胞内Ca~(2+)分布区域和浓度的变化,这是将细胞外重力刺激转换为细胞内化学信号的关键步骤.由于机械力敏感的C~(2+)通道的活化和Ca~(2+)-ATPase酶活性受到抑制,重力改变时细胞质中自由Ca~(2+)浓度增加,随后Ca~(2+)作为第二信使介导相关酶活性发生改变,最终引起一连串的生理生化反应.本文探讨了重力变化对植物细胞质内自由Ca~(2+)浓度的影响、Ca~(2+)信号的产生机制.以及Ca~(2+)作为次级信号对细胞生理生化过程调节作用的途径和机制,介绍了常用的Ca~(2+)研究方法,并分析了研究的关键点和难点.     

LED光谱对模拟空间培养箱中植物生长发育的影响

通过研究在空间植物培养箱中利用LED作为光源对植物生长发育的作用, 并以荧光灯作为对照, 评估LED光源在空间植物培养中的优缺点, 可为中国即将在空间实验室天宫二 号和空间站中开展的高等植物生长实验提供参考. 利用不同比例的红光与白光LED组合光源, 研究光谱组成(红蓝光比例)、光照强度、光周期和气体流通等条件对于模拟空间 植物培养箱中拟南芥和水稻生长发育的影响. 结果表明, 与荧光灯相比, 红蓝光比例高的LED会导致拟南芥提早开花和水稻叶片的早衰. 红蓝光峰值比在3.9左右时, 拟南芥 和水稻生长最为有利; 红蓝光峰值比超过16则明显抑制拟南芥和水稻的生长, 导致叶片早衰. 另外, 在密闭培养箱中, 光强小于150μmol·m-2·s-1时, 增加光照强度可以部分抵消气体流通不足导致拟南芥植物生长的抑制, 而光照强度大 于150μmol·m-2·s-1时, 光强越大拟南芥的生长发育受到抑制越严重. 水稻对密闭培养环境中高光强的耐受性明显好于拟南芥. 因此, 在设计空间植物培养箱的LED光照系统时, 红蓝光的比例选择是关键, 此外还需综合考虑空间微重力条件下气体对流变化影响植物对光的反应.      

Inhibitory effect of simulated microgravity on differentiating preosteoblasts

The bone loss induced by microgravity is partly due to the decrease of mature osteoblasts. In the present study, we employed the random positioning machine (RPM) to simulate microgravity and investigated the acute effects of simulated microgravity on the differentiation of 2T3 preosteoblasts. Following 7 days’ culture under normal (1 g) condition, cells were exposed to simulated microgravity for 24 h. The results showed that 24 h treatment of simulated microgravity significantly decreased alkaline phosphatase (ALP) activity without changing the cell morphology. In addition, the mRNA expressions of osteogenic genes, including runt-related gene 2 (Runx2), osterix, osteocalcin (OC), type I collagen (Col I) and bone morphogenetic protein (BMP), were dramatically downregulated. Moreover, western blot analysis of total extracellular signal-regulated kinase (Erk) and phosphorylated Erk (p-Erk) indicated that p-Erk level, which represents the Erk activation status, was increased. Taken together, our results suggested that acute exposure to simulated microgravity inhibited osteoblast differentiation through modulating the expression of osteogenic genes and the Erk activity. These findings provide new insight for bone loss due to microgravity and unloading.     

The declined phosphorylation of Heat shock protein 27 in rat cardiac muscle after hindlimb unloading

Hindlimb unloading can induce the cardiac atrophy and diminished cardiac function, however, the mechanisms responsible for which remain elusive. The chronic volume unloading of heart, which decreases the local mechanical stress, may lead to cardiac atrophy after hindlimb unloading. Many studies showed that integrin signaling, p38 MAPK, Heat shock protein 27 and cytoskeleton involved in the hypertrophic growth induced by mechanical stress. However, the mechanisms responsible for cardiac atrophy after hindlimb unloading are still unclear. In this study, we used the tail-suspended, hindlimb unloading rat model to simulate the effects of microgravity. Western blot analysis was used to detect the protein expression of Heat shock protein 27, focal adhesion kinase, p38 MAPK and their phosphorylation levels in rat cardiac muscle after 14d hindlimb unloading. The results showed that the phosphorylation levels of both Heat shock protein 27 and p38 MAPK were decreased significantly in rat cardiac muscle after hindlimb unloading. However, the phosphorylation level of focal adhesion kinase was not decreased significantly. The results suggested that Heat shock protein 27, the downstream of p38 MAPK, might play a critical role in the cardiac atrophy in response to simulated microgravity induced by hindlimb unloading.     

骨特异性转录因子Runx2对抗空间骨丢失效应的初步研究

为了构建稳定过表达Runx2 (骨特异性转录因子)的C2C12 (小鼠成肌细胞)和MG63 (前成骨细胞)细胞株, 并用于研究Runx2在对抗空间骨丢失效应中的作用. 利用实时定量聚合酶链式反应鉴定Runx2下游基因I型胶原、碱性磷酸酶表达情况, 在二维回转器中培养稳定细胞株, 通过定量聚合酶链式反应,观察在模拟失重效应下Runx2基因对其下游基因表达的影响. 结果表明, 通过筛选获得稳定转染的C2C12-Runx2和MG63-Runx2细胞株, 经鉴定都能过表达Runx2. 转染后的细胞 I 型胶原和碱性磷酸酶mRNA表达增高. 回转组与对照组相比, MG63, C2C12-Runx2, MG63-Runx2细胞的I型胶原和碱性磷酸酶mRNA表达降低, 但在模拟失重效应下, 转染细胞中I型胶原和碱性磷酸酶的mRNA表达下降程度明显低于未转染细胞株. 所构建的C2C12-Runx2和MG63-Runx2细胞株比较稳定, 并证实Runx2能部分对抗失重引起的成骨特异性分子的降低.      

X-ray induced alterations in the differentiation and mineralization potential of murine preosteoblastic cells

To evaluate the effects of ionizing radiation (IR) on murine preosteoblastic cell differentiation, we directed OCT-1 cells to the osteoblastic lineage by treatment with a combination of β-glycerophosphate (β-GP), ascorbic acid (AA), and dexamethasone (Dex). In vitro mineralization was evaluated based on histochemical staining and quantification of the hydroxyapatite content of the extracellular bone matrix. Expression of mRNA encoding Runx2, transforming growth factor β1 (TGF-β1), osteocalcin (OCN), and p21^CDKN1A was analyzed. Exposure to IR reduced the growth rate and diminished cell survival of OCT-1 cells under standard conditions. Notably, calcium content analysis revealed that deposition of mineralized matrix increased significantly under osteogenic conditions after X-ray exposure in a time-dependent manner. In this study, higher radiation doses exert significant overall effects on TGF-β1, OCN, and p21^CDKN1A gene expression, suggesting that gene expression following X-ray treatment is affected in a dose-dependent manner. Additionally, we verified that Runx2 was suppressed within 24 h after irradiation at 2 and 4 Gy. Although further studies are required to verify the molecular mechanism, our observations strongly suggest that treatment with IR markedly alters the differentiation and mineralization process of preosteoblastic cells.     

Calcium influx through stretch-activated channels mediates microfilament reorganization in osteoblasts under simulated weightlessness

We have explored the role of Ca²⁺ signaling in microfilament reorganization of osteoblasts induced by simulated weightlessness using a random positioning machine (RPM). The RPM-induced alterations of cell morphology, microfilament distribution, cell proliferation, cell migration, cytosol free calcium concentration ([Ca²⁺]_i), and protein expression in MG63 osteoblasts were investigated. Simulated weightlessness reduced cell size, disrupted microfilament, inhibited cellular proliferation and migration, and induced an increase in [Ca²⁺]_i in MG63 human osteosarcoma cells. Gadolinium chloride (Gd), an inhibitor for stretch-activated channels, attenuated the increase in [Ca²⁺]_i and microfilament disruption. Further, the expression of calmodulin was significantly increased by simulated weightlessness, and an inhibitor of calmodulin, W-7, aggravated microfilament disruption. Our findings demonstrate that simulated weightlessness induces Ca²⁺ influx through stretch-activated channels, then results in microfilament disruption.     

Structure of cress root statocytes in microgravity (Bion-10 mission).

Experiments on primary roots of Lepidium sativum L. have been performed on board the Bion-10 satellite. The experimental set-up was extremely miniaturized and completely automatic. The results demonstrate the effectiveness of the instrumentation. The spatial orientation, growth, root cap differentiation and statocyte structure of roots grown under microgravity (MG) have been compared with control roots grown on the ground (GC) and in a 1G-reference centrifuge in space (RC). Root length and cap shape did not differ between MG and control samples. Under MG, the mean distance of the statoliths from the distal cell wall of the statocytes increased significantly, the mean distance of the mitochondria decreased and the nucleus did not change its position in comparison to both controls. The number and the shape of the amyloplasts (statoliths) were not influenced by the space flight factors, but their size as well as their relative area in the cell decreased. The number of starch grains per statolith as well as their size and shape changed under MG. In MG and RC samples the number of lipid bodies in the statocytes was higher and the relative area larger than in GC samples. The relative area occupied by vacuoles was greater in RC statocytes than in GC and MG statocytes. These results partly confirm and, in addition, extend the data from earlier experiments in space.     

Effects of microgravity on c-fos gene expression in osteoblast-like MC3T3-E1 cells.

The paper summarizes the data on proliferation and gravity-related gene expression of osteoblasts that were obtained from an experiment conducted under simulated and real microgravity conditions. Simulated microgravity conditions obtained in a clinostat depress proliferation of both osteoblast-like MC3T3-E1 and HeLa carcinoma cells. This depression of proliferation occurs in a collagen gel culture in which the flow of culture medium by rotation may be reduced. Interestingly, MC3T3-E1 cells which are probably one of target cells to microgravity are more sensitive than the HeLa cells. Simulated microgravity inhibited the epidermal growth factor (EGF)-induced c-fos gene expression in the MC3T3-El cells. To examine in detail the effect of real microgravity on the EGF signal transduction cascade in osteoblasts, MC3T3-E1 cells were cultured in the Cell Culture Experiment Module of the sounding rocket TR-1A6. The EGF-induced c-fos expression in cells was depressed under short-term microgravity conditions in the sounding rocket, while the phosphorylation of mitogen-activated protein kinase (MAPK) was not affected compared with the controls grown on the ground. These results suggest that an action site of microgravity in the signal transduction pathway may be downstream of MAPK.     

Function of the cytoskeleton in gravisensing during spaceflight.

Since astronauts and cosmonauts have significant bone loss in microgravity we hypothesized that there would be physiological changes in cellular bone growth and cytoskeleton in the absence of gravity. Investigators from around the world have studied a multitude of bone cells in microgravity including Ros 17/2.8, Mc3T3-E1, MG-63, hFOB and primary chicken calvaria. Changes in cytoskeleton and extracellular matrix (ECM) have been noted in many of these studies. Investigators have noted changes in shape of cells exposed to as little as 20 seconds of microgravity in parabolic flight. Our laboratory reported that quiescent osteoblasts activated by sera under microgravity conditions had a significant 60% reduction in growth (p<0.001) but a paradoxical 2-fold increase in release of the osteoblast autocrine factor PGE2 when compared to ground controls. In addition, a collapse of the osteoblast actin cytoskeleton and loss of focal adhesions has been noted after 4 days in microgravity. Later studies in Biorack on STS-76, 81 and 84 confirmed the increased release of PGE2 and collapse of the actin cytoskeleton in cells grown in microgravity conditions, however flown cells under 1 g conditions maintained normal actin cytoskeleton and fibronectin matrix. The changes seen in the cytoskeleton are probably not due to alterations in fibronectin message or protein synthesis since no differences have been noted in microgravity. Multiple investigators have observed actin and microtubule cytoskeletal modifications in microgravity, suggesting a common root cause for the change in cell architecture. The inability of the O g grown osteoblast to respond to sera activation suggests that there is a major alteration in anabolic signal transduction under microgravity conditions, most probably through the growth factor receptors and/or the associated kinase pathways that are connected to the cytoskeleton. Cell cycle is dependent on the cytoskeleton. Alterations in cytoskeletal structure can block cell growth either in G1 (F-actin microfilament collapse), or in G2/M (inhibition of microtubule polymerization during G2/M-phase). We therefore hypothesize that microgravity would inhibit growth in either G1, or G2/M.     

Effects of heavy ions on visual function and electrophysiology of rodents: the ALTEA-MICE project.

ALTEA-MICE will supplement the ALTEA project on astronauts and provide information on the functional visual impairment possibly induced by heavy ions during prolonged operations in microgravity. Goals of ALTEA-MICE are: (1) to investigate the effects of heavy ions on the visual system of normal and mutant mice with retinal defects; (2) to define reliable experimental conditions for space research; and (3) to develop animal models to study the physiological consequences of space travels on humans. Remotely controlled mouse setup, applied electrophysiological recording methods, remote particle monitoring, and experimental procedures were developed and tested. The project has proved feasible under laboratory-controlled conditions comparable in important aspects to those of astronauts' exposure to particle in space. Experiments are performed at the Brookhaven National Laboratories [BNL] (Upton, NY, USA) and the Gesellschaft für Schwerionenforschung mbH [GSI]/Biophysik (Darmstadt, FRG) to identify possible electrophysiological changes and/or activation of protective mechanisms in response to pulsed radiation. Offline data analyses are in progress and observations are still anecdotal. Electrophysiological changes after pulsed radiation are within the limits of spontaneous variability under anesthesia, with only indirect evidence of possible retinal/cortical responses. Immunostaining showed changes (e.g. increased expression of FGF2 protein in the outer nuclear layer) suggesting a retinal stress reaction to high-energy particles of potential relevance in space.