模拟失重对大肠杆菌K12基因表达及表型的影响

【目的】通过低剪切力模拟失重(Low-shear modeled microgravity,LSMMG)连续传代培养大肠杆菌,检测大肠杆菌在模拟失重条件下的表型变化及基因改变。【方法】利用旋转细胞培养系统模拟失重环境对大肠杆菌K12进行连续传代培养,对菌株进行增殖速率、耐酸性和生物膜形成的测定,以此评估LSMMG对大肠杆菌K12表型的影响。利用转录组测序检测模拟失重条件下差异表达的基因,与表型作比对。【结果】模拟失重导致大肠杆菌增殖速率降低,耐酸性下降,生物膜形成能力增强;模拟失重条件下,营养代谢相关差异表达基因有25个,其中20个表达下降,2个与耐酸相关基因表达均下降。【结论】模拟失重会引起大肠杆菌表型及相应的基因变化,其中生物膜形成能力的增强可能对航天飞行造成潜在威胁。     

模拟失重环境对大肠杆菌K12表型异质性亚群菌株的影响

【背景】我国未来几年深空探索任务将呈"井喷式"发展,微生物对于航天活动的影响越来越引起关注,而国内外少有表型异质性亚群的研究。【目的】从表型异质性的角度探讨低剪切力模拟失重环境(Low-shearmodeledmicrogravity,LSMMG)和低剪切力正常重力环境(Low-shearnormalgravity,LSNG)对大肠杆菌K12造成的影响。【方法】利用旋转细胞培养系统模拟失重环境对大肠杆菌K12进行连续传代培养,从单克隆形态、颜色以及菌体形态等方面挑选出表型异质性的亚群菌株,对不同菌株进行增殖速率、抗生素耐药性、生物被膜形成、环境压力抵抗力以及细胞毒性的测定,以此评估低剪切力和模拟失重环境对大肠杆菌K12的影响。【结果】利用旋转细胞培养系统连续传代培养,总共分离出4株形态不同的表型异质性亚群菌株,其中2株来自模拟失重组(M1,Ma),另外2株来自正常重力对照组(N1,Na);4株亚群与原始菌株(P)相比,在增殖速率、生物被膜形成、环境压力抵抗力和细胞毒性方面均有增强或减弱的明显变化,对于抗生素的耐药性无明显变化。【结论】低剪切力模拟失重环境以及存在低剪切力的正常重力环境均能引起大肠杆菌表型异质性变化,与原始菌株相比,表型异质性亚群菌株在分化上并没有统一的方向,但仍需警惕那些可能对人类造成危害的变化表型。     

白假丝酵母菌生物膜的研究进展

随着医疗水平的不断发展,越来越多的医疗操作、医疗设备和药物可能导致人体正常的微生物平衡被打破,使得机会致病菌白假丝酵母菌的感染呈现逐年上升的趋势。白假丝酵母菌在宿主或医疗器械表面形成生物膜的能力是一个十分关键的毒力因素。生物膜可以帮助白假丝酵母菌成功逃避宿主免疫并产生较强的耐药性,从而导致难治性真菌感染。本文从白假丝酵母菌生物膜的形成过程、生物膜相关的主要基因和影响生物膜毒力的因素3个方面介绍近年来的研究进展,为进一步研究白假丝酵母菌生物膜的形成机制提供参考。     

大黄素对白假丝酵母菌抑菌作用的体外实验研究

目的研究大黄素对白假丝酵母菌的活性及其产生的生物膜的抑菌作用。方法将滴有不同浓度的大黄素药液的滤纸片放在长有白假丝酵母菌的固体培养基上,观察抑菌效果的区别。在体外建立白假丝酵母菌生物膜模型,用浓度为2.500、1.250、0.625、0.312和0.165mg/mL的大黄素溶液分别作用于已建立好的白假丝酵母菌生物膜模型,观察5个浓度的大黄素对白假丝酵母菌的抑菌效果。利用激光共聚焦显微镜(CLSM)观察各个浓度下的生物膜。结果随着大黄素浓度的增加,抑菌环直径增大,抑菌率升高。CLSM观察:大黄素作用于白假丝酵母菌生物膜,使生物膜活性降低。结论大黄素对白假丝酵母菌及其生物膜有抑制作用。     

伊犁黑蜂蜂胶对不同生长状态白假丝酵母菌抑菌作用的体外研究

目的探究伊犁黑蜂蜂胶对白假丝酵母菌游离状态及其生物膜状态的影响。方法采用NCCLS M27-A2酵母微量稀释法结合平板法测定蜂胶对游离状态下白假丝酵母菌的最小抑菌浓度(MIC)和最低杀菌浓度(MBC);玻片法在体外构建白假丝酵母菌生物膜模型,采用MTT法检测蜂胶对白假丝酵母菌生物膜状态下的最低生物膜清除浓度(MBEC)及抑制作用;激光共聚焦显微镜(CLSM)观察不同浓度蜂胶醇提取物对同一时间白假丝酵母菌生物膜的抑菌效果,并进行红蓝荧光染色定量分析。结果伊犁黑蜂蜂胶对浮游白假丝酵母菌的MIC为4mg/mL,MBC为8mg/mL;伊犁黑蜂蜂胶对生物膜状态下白假丝酵母菌的MBEC为16mg/mL,随着药物浓度的增加抑菌性逐渐增大,差异具有统计学意义(P0.05);CLSM观察,伊犁黑蜂蜂胶作用于白假丝酵母菌生物膜后可使生物膜内活菌比例降低,生物膜活性减弱,生物膜内实验菌死亡率随蜂胶浓度增加呈上升趋势,与阴性对照组比较差异有统计学意义(P0.05)。结论伊犁黑蜂蜂胶在体外对不同状态下的白假丝酵母菌均起到了明显的抑制作用。     

土槿乙酸对白假丝酵母菌生物膜的抑制作用

目的探讨土槿乙酸(pseudolaric acid B,PAB)对体外白假丝酵母菌生物膜的影响。方法甲基四氮盐(XTT)法检测不同浓度PAB和AMB(两性霉素B)对白假丝酵母菌生物膜的抑制作用。血清芽管试验检测不同浓度PAB对芽管生成的影响。结果 PAB对白假丝酵母菌生物膜的SMIC50(抑制生物膜50%的药物浓度)为256~512μg/m L;1024和512μg/m L PAB对早期2 h生物膜的抑制率分别为(99.5±0.28)%和(97.1±0.38)%;512μg/m L PAB对早期(2 h)、中期(8 h)及成熟期(24 h)生物膜的抑制率分别为(97.1±0.38)%、(90.4±0.32)%和(80.1±0.67)%;不同浓度PAB的血清芽管试验显示,64μg/m L PAB可以完全抑制白假丝酵母菌的出芽生长,16μg/m L PAB可以抑制83.5%的白假丝酵母菌出芽生长。结论 PAB对体外白假丝酵母菌生物膜有抑制作用,对白假丝酵母菌的出芽生长过程抑制作用显著。     

白假丝酵母菌突变株ORF19.2500的功能研究

目的筛选50株白假丝酵母菌基因缺失菌,寻找出对白假丝酵母菌生物被膜形成相关基因,进一步探究白假丝酵母菌生物膜的致病机制。方法利用培养生物被膜的方法筛选50株基因缺失菌;利用XTT法验证所筛选出的白假丝酵母菌突变株ORF19.2500生物被膜形成缺陷;进一步观察ORF19.2500基因缺失菌生长、菌丝形成。结果用XTT法证明白假丝酵母菌突变株orf19.2500生物膜形成缺陷,且生长曲线和滴琼脂平板的方法均提示其生长速率减慢。在spider培养基上白假丝酵母菌突变株orf19.2500不能诱导菌丝形成,但在YPD+10%小牛血清则菌丝形成正常。结论白假丝酵母菌突变株orf19.2500可利用spider培养基缺陷而影响其酵母、菌丝二态性的转化,及其生物被膜的形成。     

白假丝酵母菌滞留菌形成相关机制的研究进展

白假丝酵母菌属于临床侵袭性条件致病菌,可引起从浅表黏膜到危及生命的全身感染性疾病,其形成的生物膜是为生物膜内细胞提供结构支架和保护模式的结构化菌体群落,其产生的滞留菌是一种随机且对抗真菌药物高度耐受的细胞亚群。由于生物膜与滞留菌两种因素的存在,致使临床治疗侵袭性真菌感染时面临极大挑战,因此,研究白假丝酵母菌滞留菌形成机制对目前临床治疗侵袭性真菌感染具有重要意义。本文对白假丝酵母滞留菌形成的主要调控因素(生物膜的形成、氧化应激反应、蛋白酶系统、TOR-RAS-CAMP-PKA信号转导途径和菌种自身因素),以及滞留菌与临床疾病的相关性进行综述。     

穿心莲内酯对白假丝酵母菌菌丝的影响

目的探讨中药有效成分穿心莲内酯（Andrographolide,AG）对白假丝酵母菌菌丝的影响。方法利用稀释涂布法观察AG对固体培养基上白假丝酵母菌菌落形态的影响;倒置显微镜观察AG对白假丝酵母菌芽管及菌丝形态的影响;采用xTT还原法评价AG对白假丝酵母菌菌丝活性的影响;采用实时荧光定量PCR（qRT—PCR）检测白假丝酵母菌菌丝形成相关基因SUN41、CSHl以及CaPDE2表达量变化。结果AG能影响白假丝酵母菌菌落的形态;倒置显微镜观察表明AG能够抑制白假丝酵母菌菌丝及芽管的形成;qRT-PCR检测显示AG能使SUN41和CSHl基因表达下调和使CaPDE2上调。结论穿心莲内酯可能通过影响SUN41、CSHl与CaPDE2基因的表达而抑制白假丝酵母菌菌丝的形成。     

纳豆杆菌对白假丝酵母菌的拮抗作用

目的探讨纳豆杆菌对白假丝酵母菌的拮抗作用。方法将纳豆杆菌和白假丝酵母菌混合培养24 h后,应用沙保弱平板培养基分离白假丝酵母菌,计数菌落,计算纳豆杆菌对白假丝酵母菌的拮抗率。结果纳豆杆菌对白假丝酵母菌的拮抗作用明显,拮抗率高达91.91%;纳豆杆菌肉汤培养物的除菌滤液对白假丝酵母菌也有明显的拮抗作用,拮抗率为79.05%。结论纳豆杆菌对白假丝酵母菌具有明显拮抗作用,是白假丝酵母菌的理想拮抗菌株。     

Microarray analysis of genes differentially expressed in hepG2 cells cultured in simulated microgravity: Preliminary report

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.     

Effects of chronic exposure to simulated microgravity on skeletal muscle cell proliferation and differentiation

Cell culture models that mimic long-term exposure to microgravity provide important insights into the cellular biological adaptations of human skeletal muscle to long-term residence in space. We developed insert scaffolding for the NASA-designed rotating cell culture system (RCCS) in order to study the effects of time-averaged microgravity on the proliferation and differentiation of anchorage-dependent skeletal muscle myocytes. We hypothesized that prolonged microgravity exposure would result in the retardation of myocyte differentiation. Microgravity exposure in the RCCS resulted in increased cellular proliferation. Despite shifting to media conditions promoting cellular differentiation, 5 d later, there was an increase in cell number of approximately 62%, increases in total cellular protein (52%), and cellular proliferating cell nuclear antigen (PCNA) content (2.7 times control), and only a modest (insignificant) decrease (10%) in sarcomeric myosin protein expression. We grew cells in an inverted orientation on membrane inserts. Changes in cell number and PCNA content were the converse to those observed for cells in the RCCS. We also grew cells on inserts at unit gravity with constant mixing. Mixing accounted for part, but not all, of the effects of microgravity exposure on skeletal muscle cell cultures (53% of the RCCS effect on PCNA at 4-6 d). In summary, the mechanical effects of simulated microgravity exposure in the RCCS resulted in the maintenance of cellular proliferation, manifested as increases in cell number and expression of PCNA relative to control conditions, with only a modest reciprocal inhibition of cellular differentiation. Therefore, this model provides conditions wherein cellular differentiation and proliferation appear to be uncoupled.     

Phenotypic characterization of Aspergillus niger and Candida albicans grown under simulated microgravity using a three-dimensional clinostat

The living and working environments of spacecraft become progressively contaminated by a number of microorganisms. A large number of microorganisms, including pathogenic microorganisms, some of which are fungi, have been found in the cabins of space stations. However, it is not known how the characteristics of microorganisms change in the space environment. To predict how a microgravity environment might affect fungi, and thus how their characteristics could change on board spacecraft, strains of the pathogenic fungi Aspergillus niger and Candida albicans were subjected to on-ground tests in a simulated microgravity environment produced by a three-dimensional (3D) clinostat. These fungi were incubated and cultured in a 3D clinostat in a simulated microgravity environment. No positive or negative differences in morphology, asexual reproductive capability, or susceptibility to antifungal agents were observed in cultures grown under simulated microgravity compared to those grown in normal earth gravity (1 G). These results strongly suggest that a microgravity environment, such as that on board spacecraft, allows growth of potentially pathogenic fungi that can contaminate the living environment for astronauts in spacecraft in the same way as they contaminate residential areas on earth. They also suggest that these organisms pose a similar risk of opportunistic infections or allergies in astronauts as they do in people with compromised immunity on the ground and that treatment of fungal infections in space could be the same as on earth.     

Microgravity effect on testicular functions

In mammals spaceflight influences spermatogenesis since spermatogonial germ cell proliferation, compared to synchronous controls, is lightly decreased in irradiated or flown rats. Moreover, changes of the plasmatic testosterone production was described either in flight rats, or in rats maintained in simulated microgravity conditions. The hormonal levels of the astronauts change as it has been previously described, including hormones involved in the regulation of spermatogenesis such as testosterone and luteinizing hormone (LH). In microgravity conditions, human testosterone levels decreased whereas circulating LH levels increased. To study the effect of simulated microgravity on mammalian spermatogenesis we have utilized the Rotary Cell Culture System (RCCS) and we have cultured testicular fragments isolated from prepuberal rats in a chemically defined medium for three days under microgravity conditions. As control we have cultured the same amount of fragments at unit gravity. The morphology of the samples has been studied and the number of proliferating cells has been counted in control samples and in samples maintained in RCCS. The results indicate that the number of duplicating cells in the tubules was significantly increased in the microgravity-cultured fragments. The amount of testosterone secreted in the culture medium has been also evaluated and in RCCS samples the amount of the hormone was higher respect to the control samples.     

Induction of three-dimensional assembly of human liver cells by simulated microgravity

Summary The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.     

结肠癌中核内miRNA的激活调控作用研究

为了探究增强子介导的核内miRNA在结肠癌发生中的作用,本研究筛选了结肠癌中的差异表达的miRNA数据、结肠的特异性增强子数据、结肠癌中差异表达基因数据,利用细胞核内miRNA靶向增强子预测算法,筛选miRNA调控的结肠特异性增强子;利用增强子靶基因预测数据,筛选核内miRNA调控的差异表达靶基因,并且构建核内miRNA-靶基因网络,并通过网络的分析和筛选获得结肠癌中关键的致病基因,同时对网络中的靶基因进行GO的功能注释。结果表明,我们所构建的核内miRNA-激活调控靶基因网络包含miRNA-靶基因关系对2 121个,259个节点,其中包含34个下调基因、183个上调的基因,7个下调的miRNA,35个上调的miRNA。而后我们分析了网络进行的节点度的整体分布情况,发现网络中大部分的节点的度都是小于10的,仅有少量miRNA结合和部分的差异表达基因节点的度大于10。核内miRNA主要通过激活调控了一些应激反应相关的功能和,同时,抑制调控了细胞周期、细胞凋亡、细胞死亡巨噬细胞代谢等相关功能,通过激活和抑制相关功能诱发结肠癌的发生。从核内miRNA的激活调控角度研究结肠癌的发病机制,是对原有细胞浆中miRNA抑制调控机制的补充,也为结肠癌的系统研究提供了新的视野。