一种新的在线细胞显微观察仪及其在生化反应器上的应用研究

针对生化反应器应用条件,提出了用于生化反应器的在线细胞观察仪的基本技术要求。在比较了国内外现有的细胞在线显微工作原理后,研制了一种基于暗视场的新的显微细胞观察仪,介绍了其关键技术及结构。另外,原位在线显微细胞观察仪应用于酿酒酵母和哺乳动物细胞HEK293细胞的培养试验,在线细胞计数结果与离线细胞计数和细胞干重相比较,均有很好的相关性,表明此仪器基本满足使用要求。     

新的酵母细胞自动计数仪

BioScience Technology 05／2005（增刊）10页报道：美国新不伦瑞克科学公司最近推出了一种可以在30秒内自动计数酵母细胞的新的仪器,从而结束了手工计数酵母细胞的历史。与原先可以自动计数哺乳动物细胞的核计数仪相似,这种新的可用于计数酵母细胞的YC-100型核计数仪,包含荧光显微镜、CCD照相机和整合影像分析软件,可快速而准确地对酵母细胞进行计数。它可以根据DNA在数秒钟内进行细胞计数,     

“细胞的结构与功能-Ⅰ”的教学组织

细胞是生命的基本单位。细胞很小,却被称为复杂性巨大的微缩奇观。细胞生物学研究分3个层次：显微水平、亚显微水平和分子水平．它是一切生命科学研究的基础,即一切生命现象都要从细胞中获得答案。关于细胞的结构与功能的教学组织,本文重点探讨2方面内容：一是细胞学说的建立;二是细胞膜的结构与功能。生物学新课程标准提出的具体要求是：分析细胞学说建立的过程：使用显微镜观察多种多样的细胞：简述细胞膜系统的结构与功能。     

小胶质细胞纯化分离培养方法的改良

目的改良现有的小胶质细胞纯化分离培养方法,建立稳定简便的培养模型。方法利用盐酸利多卡因注射液代替机械振摇纯化分离小胶质细胞;利用CD11b/c(OX42)免疫细胞化学的方法对分离的小胶质细胞纯度进行鉴定,同时观察小胶质细胞形态及活化指标NFкBp65的表达情况;利用流式细胞仪,结合细胞计数及MTT细胞活力测定检测纯化分离后小胶质的增殖情况。结果改良的方法可稳定的获得1.2×106个/培养瓶(75cm2,250ml)的小胶质细胞,纯度达到98%,存活率≥95%,形态上以阿米巴样为主,继续培养3-5d,约半数细胞可转变为静止状态。NFкBp65免疫细胞化学染色为胞浆表达。流式细胞仪检测结合细胞计数及MTT细胞活力检测结果显示,体外纯化培养的小胶质细胞多位于G0/G1期,培养过程中未出现明显的增殖。结论改良的方法易于操作,产量多,纯度高。为体外小胶质细胞进一步研究提供了基础。     

过表达Nogo-C对PC12细胞存活及增殖的影响

以PC12细胞为神经元细胞模型,研究Nogo-C对神经元细胞存活及增殖的作用。在PC12细胞中转染过表达Nogo-C,使用G418药物筛选以获得稳定表达的细胞克隆,利用Hoechst33342染色、细胞计数、MTT以及流式细胞仪等技术检测Nogo-C对细胞增殖以及细胞周期的影响。结果表明:(1)Hoechst33342染色未观察到表达Nogo-C的细胞发生明显凋亡;(2)细胞计数及MTT实验观察到转染Nogo-C后的PC12细胞生长增殖活性明显降低;(3)流式细胞仪检测细胞生长周期,正常PC12细胞G1期的百分数为(37.8±7.9)%,S期为(50.4±8.5)%,而转染Nogo-C的PC12细胞G1期为(76.8±4.1)%,S期为(14.7±1.7)%,提示转染Nogo-C的PC12细胞的细胞周期被阻滞在G1期;(4)没有获得稳定表达Nogo-C的PC12细胞模型。实验证明,过表达Nogo-C通过使PC12细胞周期被阻滞在G1期而明显抑制细胞的增殖,但是并不引起细胞的凋亡。     

绿僵菌素A和B对斜纹夜蛾SL-1细胞的增殖抑制和致凋亡作用

绿僵菌素A和B是生物活性分子,本研究用MTT法、相差显微观察、荧光倒置显微观察和流式细胞术比较了绿僵菌素A和B对斜纹夜蛾Spodoptera litura SL-1细胞的毒杀作用。结果表明：绿僵菌素A和B对SL-1细胞均具有明显的增殖抑制作用,且具有正比的时间-浓度-效应关系,绿僵菌素A和B处理细胞48 h后的IC50值分别为7.80和20.73 μg/mL。倒置相差显微观察发现,绿僵菌素A和B可以引起SL-1细胞变圆、胞膜收缩、有凋亡小颗粒形成。随着时间的延长,细胞悬浮致死并出现大量空泡和胞质外泄现象。但是在同样的处理浓度下,绿僵菌素A的作用较绿僵菌素B明显。用AO/EB染色后,荧光显微观察发现：绿僵菌素A 诱导的细胞荧光强度高于绿僵菌素B。流式细胞仪检测结果表明：绿僵菌素A和B对SL-1细胞具有明显的致凋亡作用。10 μg/mL绿僵菌素A和B作用细胞48 h后,SL-1细胞的总凋亡率分别达78.88%±0.97%和72.23%±2.29%。本研究从细胞水平肯定了绿僵菌素具有良好的增殖抑制和致凋亡作用,并且为它在害虫防治中的潜在应用提供了一些理论支持。     

膜过滤细胞直接计数新方法

介绍了两种膜过滤细胞直接计数新方法,即不透明滤膜的金相显微镜细胞直接计数法和透明滤膜的生物显微镜细胞直接计数法的基本原理、仪器设备、分析和计算方法,并且将此方法与荧光显微镜细菌计数、血球计数板等方法进行了比较。实际计数结果表明,两种膜过滤细胞直接计数法配合了独特的酸碱处理法以分散聚团细胞,用于非环境样品细胞浓度的测定具有快速准确简便的特点。此外,该方法基本不受细胞大小的影响,除了适用于体型较大的藻类、酵母菌的快速计数以外,还可以清晰地分辨诸如光合细菌这样体积较小的细菌。     

细胞计数板在荧光显微镜观察B细胞吞噬现象的应用探讨

目的:基于细胞计数板建立一种简单、快速使用免疫荧光显微镜观察B淋巴细胞吞噬卡介苗(BCG)现象的新方法,对即将进行流式细胞检测的样品进行质控,提高流式细胞术检测吞噬率的稳定性,同时为流式细胞仪检测吞噬率提供镜下依据。方法:B细胞与FITC标记的BCG共培养24 h后,PE anti-human CD19抗体直接标记细胞膜,应用细胞计数板在荧光显微镜下观察B细胞吞噬现象,流式细胞仪检测吞噬率。结果:应用细胞计数板在荧光镜下可观察到B细胞与BCG的荧光标记及B细胞与BCG共标记现象,证实B细胞可吞噬BCG,流式细胞仪检测结果显示吞噬率为13.9%。结论:应用细胞计数板在荧光镜下可观察B细胞吞噬现象,且操作简便快速,能对流式细胞检测的样品进行质控,并提供镜下依据。     

电容法在线测定发酵过程中毕赤酵母浓度的研究

活细胞量是与细胞生长、代谢、生产力相关的重要生理参数。在线活细胞测定仪能够利用电容的原理检测发酵液中的活细胞量。本文详细介绍了国产在线活细胞测定仪的检测原理,并说明了其在毕赤酵母发酵过程中的应用情况。结果表明,我们设计的在线活细胞测定仪能够直接、实时、在线获得发酵过程中可靠的活细胞量,且有效避免采样操作的干扰。这一技术符合过程分析技术(PAT)的要求,并可以与其他参数相结合进行分析,作为发酵过程开发、控制和优化的有利工具。     

《分子与细胞生物学期刊》：细胞环境对p53分子调控网络影响机制研究获进展

《分子与细胞生物学期刊》（JournalofMolecularCellBiology）近日在线发表了中科院生化与细胞所吴家睿研究组与陈洛南研究组共同合作取得的研究成果,报道了一种细胞环境对p53分子调控网络的影响机制及其在肿瘤发生中的作用。     

Mass transfer analysis for immobilized cells of Lactococcus lactis sp. using both simulations and in-situ pH measurements

Mathematical modeling and in-situ pH measurements were used to characterize the effects of the microenvironment on alginate gel beads immobilized cells of Lactococcus lactis. Mass transfer limitations led to a progressive pH acidification within gel beads which determined both the cell distribution and the cellular activity of entrapped cells. The dynamics of the system is discussed in relation to the overall activity of the immobilized cell reactor.     

Some aspects of hybridoma cell cultivation

Summary Two hybridoma cell lines were cultivated in an indirectly aerated 10-1 reactor in batch, fed-batch and continuous (perfusion) operations and in spinner flasks. The medium in the reactor was sampled either by an aseptic cross-flow filtration module integrated into a loop or by an in-situ tubular filter. The glucose concentration was monitored by an on-line flow injection analyzer and the ammonia concentration by an ion-selective electrode. Since the membrane transmission of the high-molecular components decreased during cultivation, the product, a monoclonal antibody, was enriched in the reactor. During cultivation, the concentrations of cells, viable cells, glucose, lactase, acetate, citrate, ammonia, urea, amino acids, proteins, and monoclonal antibodies were determined off-line. The specific growth rate, specific production, and consumption rates of the medium components were influenced considerably by the medium composition, especially by the type and amount of serum used.Offprint requests to: K. Schügerl     

Anaerobic treatment of wastewater with high suspended solids from a bulk drug industry using fixed film reactor (AFFR)

Studies were carried out on the treatment of wastewater from a bulk drug industry using an anaerobic fixed film reactor (AFFR) designed and fabricated in the laboratory. The chemical oxygen demand (COD) and total dissolved solids (TDS) of the wastewater were found to be very high with low biochemical oxygen demand (BOD) to COD ratio and high total suspended solid (TSS) concentration. Acclimatization of seed consortia and startup of the reactor was carried out by directly using the wastewater, which resulted in reducing the period of startup to 30 days. The reactor was studied at different organic loading rates (OLR) and it was found that the optimum OLR was 10 kg COD/m(3)/day. The wastewater under investigation, which had a considerable quantity of SS, was treated anaerobically without any pretreatment. COD and BOD of the reactor outlet wastewater were monitored and at steady state and optimum OLR 60-70% of COD and 80-90% of BOD were removed. The reactor was subjected to organic shock loads at two different OLR and the reaction could withstand the shocks and performance could be restored to normalcy at that OLR. The results obtained indicated that AFFR could be used efficiently for the treatment of wastewater from a bulk drug industry having high COD, TDS and TSS.     

Bubble bed reactor: A reactor design to minimize the damage of bubble aeration on animal cells

A new bubble aeration system was designed to minimize cell killing and cellular damage due to sparging. The residence time of the bubbles in the developed bubble bed reactor was prolonged dramatically by floating them in a countercurrent produced by an impeller. The performance of the new reactor bubble aeration system, implemented in a laboratory reactor, was tested in dynamic aeration experiments with an without cells. An efficiency up to 95% in oxygen transfer could be achieved, which enables a much lower gas flow rate compared with conventional bubble aeration reactors. The low gas flow rate is important to keep cell damage by bubbles as low as possible. A laser light sheet technique used to find the optimal flow pattern in the reactor. The specific power dissipation of the impeller is a good measure to predict cell damage in a turbulent flow. Typical values for the power dissipation measured in the bubble bed reactor were in the range of 0.002 to 0.013 W/kg, which is far below the critical limit for animal cells. The growth of a hybridoma cell line was studied in cell cultivation experiments. A protein-free medium without supplements such as serum or Pluronic F68 was used to exclude any effect of cell-protecting factors, No difference in the specific growth rate and the yield of the antibodies was observed in cell grown in the bubble free surface aeration in the spinner flask. In contrast to the spinner flask, however, the bubble bed reactor design could be scaled up. (c) 1994 John Wiley & Sons, Inc.     

激光共聚焦显微镜在微痕定量分析中的应用综述

微痕分析是石器、骨器、牙齿等工艺、功能研究的常用方法,但无论是高倍还是低倍等常用方法,受限于体式光学显微镜、扫描电镜等观察方法,微痕分析通常很难进行原位定量分析,因此微痕分析的定量化是目前学术界遇到的主要问题。近年来随着激光共聚焦显微镜（LSCM）在微痕分析中的应用,微痕定量化在西方学术界得到了新的实践和进展。本文主要介绍激光共聚焦显微镜的原理及其在微痕分析中的应用案例,以期促进微痕分析在中国的进一步发展。     

锰对PC12 细胞的增殖抑制与凋亡研究

目的:研究锰作用下PC12细胞的增殖抑制作用与凋亡相关的形态学、生化指标改变。方法:用200,400,600,800μmol/LMnCl2的培养液,分别作用对数生长期PC12细胞1,2,3,4d后,用MTT筛选锰的细胞毒性剂量;透射电镜观察细胞形态学变化;琼脂糖凝胶电泳检测MnCl2对PC12细胞基因组DNA的影响。结果:MTT实验显示200-800μmol/L MnCl2作用4天对PC12有显著的抑制作用,呈剂量和时间依赖趋势,600μmol/L MnCl2作用4d对PC12的抑制率可达50%以上。600μmol/L MnCl2作用4d电镜可见细胞凋亡,同样条件下细胞DNA碎片化。结论:PC12细胞在锰作用下发生增殖抑制,原因是锰诱导PC12细胞凋亡。     

Nutrient-split feeding strategy for dialysis cultivation of Escherichia coli

Reduction in nutrient loss during dialysis cultivation of Escherichia coli on a glycerol medium was investigated. A dialysis reactor with an inner fermentation and an outer dialysis chamber was used. Aerobic condition was maintained by limiting the glycerol feed rate to an optimum value which was estimated from the oxygen requirements for glycerol oxidation and oxygen transfer capacity of the reactor. High reduction in nutrient loss was achieved by using water as the dialyzing fluid. However, osmotic movement of water from the dialysis to the fermentation chamber was observed, and the final cell concentration was low. With a nutrient-split feeding strategy (feeding glycerol directly to the fermentation chamber and dialyzing with salt solution), glycerol loss was small, there was no osmotic flux of water to the fermentation chamber, and the cell concentration was high. Both glycerol and salt loss could be avoided, and a cell concentration of 170 g/L was obtained when the dialysis process was substituted by addition of XAD adsorbents to the dialysis chamber. Application of this nutrient-split feeding strategy to cell cultivation in a stirred tank reactor, coupled with dialysis in external dialyzer modules, resulted in low cell concentrations. (c) 1993 Wiley & Sons, Inc.     

A framework for whole-cell mathematical modeling

The default framework for modeling biochemical processes is that of a constant-volume reactor operating under steady-state conditions. This is satisfactory for many applications, but not for modeling growth and division of cells. In this study, a whole-cell modeling framework is developed that assumes expanding volumes and a cell-division cycle. A spherical newborn cell is designed to grow in volume during the growth phase of the cycle. After 80% of the cycle period, the cell begins to divide by constricting about its equator, ultimately affording two spherical cells with total volume equal to twice that of the original. The cell is partitioned into two regions or volumes, namely the cytoplasm (Vcyt) and membrane (Vmem), with molecular components present in each. Both volumes change during the cell cycle; Vcyt changes in response to osmotic pressure changes as nutrients enter the cell from the environment, while Vmem changes in response to this osmotic pressure effect such that membrane thickness remains invariant. The two volumes change at different rates; in most cases, this imposes periodic or oscillatory behavior on all components within the cell. Since the framework itself rather than a particular set of reactions and components is responsible for this behavior, it should be possible to model various biochemical processes within it, affording stable periodic solutions without requiring that the biochemical process itself generates oscillations as an inherent feature. Given that these processes naturally occur in growing and dividing cells, it is reasonable to conclude that the dynamics of component concentrations will be more realistic than when modeled within constant-volume and/or steady-state frameworks. This approach is illustrated using a symbolic whole cell model.     

Optical aberrations and objective choice in multicolor confocal microscopy

Refinements in design have simplified confocal microscopy to the extent that it has become a standard research tool in cell biology. However, as confocal microscopes have become more powerful, they have also become more demanding of their optical components. In fact, optical aberrations that cause subtle defects in image quality in wide-field microscopy can have devastating effects in confocal microscopy. Unfortunately, the exacting optical requirements of confocal microscopy are often hidden by the optical system that guarantees a sharp image, even when the microscope is performing poorly. Optics manufacturers provide a wide range of microscope objectives, each designed for specific applications. This report demonstrates how the trade-offs involved in objective design can affect confocal microscopy.     

Experimental ATR device for real-time FTIR imaging of living cells using brilliant synchrotron radiation sources

In this contribution we present the design of an original Attenuated Total Reflection (ATR)-based device designed for an IR microscope coupled to a FPA detector and optimized for in-vivo cell imaging. The optical element has been designed to perform real time experiments of cell biochemical processes. The device includes a manually removable Ge-crystal that guarantees an ease manipulation during the cell culture and a large flat surface to support the cell growth and the required change of the culture wells. This layout will allow performing sequential ATR IR imaging with the crystal immersed in the culture wells, minimizing contributions due to water vapors in the optical system. Using existing brilliant synchrotron radiation sources this ATR device may collect images at the surface of the Ge crystal at a sub-cellular spatial resolution with a penetration depth of the evanescent wave inside the sample of ~ 500 nm within few seconds. A brief summary of the cellular components that should be detected with such optical device is also presented.