模拟微重力对人骨髓间充质干细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(Ruax2)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.      

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.     

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.     

太空环境对肿瘤细胞生理特性的影响

将3种肿瘤细胞搭载于“神舟4号”的卫星返回舱内,经过7天太空飞行,回收后对存活细胞进行单克隆化,观察细胞形态,并测定了细胞周期、黏附力及细胞因子表达.结果显示,经太空飞行,小鼠黑色素瘤B16细胞的周期发生改变,G1期细胞明显增多(p<0.05),并表现多种细胞形态;人肺鳞癌细胞L78对血管内皮细胞黏附力明显减弱,但经传代培养其黏附力恢复且超过对照组细胞;Caski细胞IL-2、IL-8、TNF和TGF的表达均明显增加,而L78细胞上述4种细胞因子的表达均显著下降.结论,太空环境可影响肿瘤细胞的某些生理特性,但可否影响肿瘤细胞的免疫原性,仍需做进一步的实验.     

Theoretical simulation of electron density and temperature distribution at Indian equatorial and low latitude ionosphere

The electron density and temperature distribution of the equatorial and low latitude ionosphere in the Indian sector has been investigated by simultaneously solving the continuity, momentum and energy balance equations of ion and electron flux along geomagnetic field lines from the Northern to the Southern hemisphere. Model algorithm is presented and results are compared with the electron density and electron temperature measured in situ by Indian SROSS C2 satellite at an altitude of ∼500 km within 31°S–34°N and 75 ± 10°E that covers the Indian sector during a period of low solar activity. Equatorial Ionization Anomaly (EIA) observed in electron density, morning and afternoon enhancements, equatorial trough in electron temperature have been simulated by the model within reasonable limits of accuracy besides reproducing other normal diurnal features of density and temperature.     

Molecular alterations in tumorigenic human bronchial and breast epithelial cells induced by high LET radiation.

Carcinogenesis is a multi-stage process with sequence of genetic events governing the phenotypic expression of a series of transformation steps leading to the development of metastatic cancer. In the present study, immortalized human bronchial (BEP2D) and breast (MCF-10F) cells were irradiated with graded doses of either 150 keV/micrometer alpha particles or 1 GeV/nucleon 56Fe ions. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Cell fusion studies indicated that radiation-induced tumorigenic phenotype in BEP2D cells could be completely suppressed by fusion with non-tumorigenic BEP2D cells. The differential expressions of known genes between tumorigenic bronchial and breast cells induced by alpha particles and their respective control cultures were compared using cDNA expression array. Among the 11 genes identified to be differentially expressed in BEP2D cells, three (DCC, DNA-PK and p21(CIP1)) were shown to be consistently down-regulated by 2 to 4 fold in all the 5 tumor cell lines examined. In contrast, their expressions in the fusion cell lines were comparable to control BEP2D cells. Similarly, expression levels of a series of genes were found to be altered in a step-wise manner among tumorigenic MCF-10F cells. The results are highly suggestive that functional alterations of these genes may be causally related to the carcinogenic process.     

Effects of space flight exposure on cell growth,tumorigenicity and gene expression in cancer cells

It is well recognized that harsh outer space environment, consisting of microgravity and radiation, poses significant health risks for human cells. To investigate potential effects of the space environment exposure on cancer cells we examined the biological changes in Caski cells carried by the “Shen Zhou IV” spaceship. After exposure for 7 days in spaceflight, 1440 survival subclonal cell lines were established and 4 cell lines were screened. 44F10 and 17E3 were selected because of their increased cell proliferation and tumorigenesis, while 48A9 and 31F2 had slower cytological events. Experiments with cell proliferation assay, flow cytometry, soft agar assay, tumorigenesis assay and DNA microarray analysis have shown that selected cell lines presented multiple biological changes in cell morphology, cell growth, tumorigenicity and gene expression. These results suggest that space environment exposure can make significant biological impact on cancer cells and provide an entry point to find the immunological target of tumorigenesis.     

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.     

The role of DNA repair on cell killing by charged particles.

It can be noted that it is not simple double strand breaks (dsb) but the non-reparable breaks that are associated with high biological effectiveness in the cell killing effect for high LET radiation. Here, we have examined the effectiveness of fast neutrons and low (initial energy = 12 MeV/u) or high (135 MeV/u) energy charged particles on cell death in 19 mammalian cell lines including radiosensitive mutants. Some of the radiosensitive lines were deficient in DNA dsb repair such as LX830, M10, V3, and L5178Y-S cells and showed lower values of relative biological effectiveness (RBE) for fast neutrons if compared with their parent cell lines. The other lines of human ataxia-telangiectasia fibroblasts, irs 1, irs 2, irs 3 and irs1SF cells, which were also radiosensitive but known as proficient in dsb repair, showed moderated RBEs. Dsb repair deficient mutants showed low RBE values for heavy ions. These experimental findings suggest that the DNA repair system does not play a major role against the attack of high linear energy transfer (LET) radiations. Therefore, we hypothesize that a main cause of cell death induced by high LET radiations is due to non-reparable dsb, which are produced at a higher rate compared to low LET radiations.     

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.