小鼠Kupffer细胞分离及细胞培养

目的 采用在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离Kupffer细胞（Kupffer cells,KCs）,探讨其在分离小鼠KCs的应用及其对KCs生物活性的影响.方法 根据原位灌注和梯度离心方法不同随机分为4组：无胶原酶原位灌注＋3层梯度离心组（A）、无胶原酶原位灌注＋双层梯度离心组（B）、胶原酶原位灌注＋3层梯度离心组（C）和胶原酶原位灌注＋双层梯度离心组（D）.采用F4/80（BM8）免疫染色及吞墨实验判断细胞纯度和功能、台盼蓝拒染实验判断细胞的活力,探讨不同方法KCs分离的效果及细胞活性.结果 刚分离的KCs细胞近似圆形,接种l h后收获细胞纯度较高,但细胞得率相对较低.培养4 h后KCs得率相对较高,培养28 d仍能存活.免疫荧光可显示分离的为KCs,台盼蓝染色显示各组细胞的活力均在90 %左右,在体胶原酶灌注和双层梯度离心可以增加KCs的得率,双层梯度离心法可以增加分离KCs的纯度.结论 在体胶原酶灌注对提高KCs得率较为重要,在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离小鼠KCs的的方法简便、高效、稳定,培养的KCs具有良好的细胞生物学性状.     

大鼠胰腺β细胞离子通道的一些特性

实验以单个Ｗｉｓｔａｒ大鼠胰腺β细胞为对象,用穿孔膜片箝和细胞贴附式记录技术研究ＡＴＰ敏感Ｋ＾＋通道（ＫＡＴＰ）、延迟整流型Ｋ＾＋通道（ＫＤＲ）、Ｃａ＾２＋通道和Ｎａ＾＋通道的有关特性。结果表明：⑴ＫＡＴＰ通道的内流电导约６５ｐＳ,外流电导约３１ｐＳ,反转电位在－６０ｍＶ左右;⑵ＫＤＲ通道在延迟２０ｍｓ后达到最大激活,ＫＤＲ电流约为ＫＡＴＰ的１／３;⑶钙电流在０ｍＶ左右达到４０－６０ｐＡ的峰值,Ｌ     

老年学习记忆减退大鼠脑突触体膜流动性改变

选用Morris水迷宫将老年大鼠分为学习记忆正常和学习记忆减退两部分,采用荧光偏振技术,对青年、老年记忆正常和老年记忆减退鼠脑分离实触体膜流动性进行测定,并检测神经节苷脂GM1对膜流动性的影响．结果表明老年记忆减退鼠新皮质、海马结构突触体膜荧光各向异性明显增加,即膜流动性显著降低,GM1对膜流动性有明显改善作用．相关分析表明新皮质、海马结构实触膜流动性与老年学习记忆减退密切相关,GM1的积极作用为临床治疗提供实验依据．     

卵泡液细胞外囊泡携带的microRNA对卵泡闭锁影响的研究进展

细胞外囊泡(extracellular vesicles, EVs)是细胞主动释放的膜结合颗粒。在原核生物和真核生物中,EVs被认为是细胞间进行信息交流的一种方式。EVs具有携带蛋白质、脂质和核酸等生物大分子的能力,可以影响亲本细胞和受体细胞的不同生理功能。其中,EVs携带的microRNA研究报道最多,在生物体生理功能方面发挥着重要作用。卵泡在发育过程中,只有少数卵泡可以充分发育、成熟、排卵,大多数卵泡在发育的不同阶段发生闭锁。在卵泡发育的整个过程中,每一阶段的变化以及卵泡闭锁调控机制还不完全清楚。本文在总结EVs类型、特性、分离方法及用途的基础上,从不同细胞因子、激素方面重点论述了卵泡液中EVs携带的microRNA是如何调控卵泡闭锁,同时对卵泡液EVs携带的microRNA在生殖调控和生殖疾病诊断方面的研究前景进行了展望,对于卵泡发育调控研究以及有效利用研究具有一定参考意义。     

卵泡液中细胞外囊泡及其携带的microRNA对卵泡发育的作用

细胞外囊泡(Extracellular vesicles,EVs)是指细胞分泌的双层膜转运囊泡。EVs能从细胞中摄取大分子物质,并将其转移至受体细胞。在这些大分子物质中,研究最多的就是microRNA (miRNA)。miRNA是一种参与基因表达调控的非编码RNA,已证实在哺乳动物卵泡液EVs中有不同的非编码RNA存在,EVs携带miRNA可以作为自分泌和旁分泌的替代机制,影响卵泡发育。文中系统介绍了EVs的种类、特征和分离鉴定方法,重点综述了EVs及携带的miRNA对卵泡发育的作用,包括早期卵泡发育、卵母细胞成熟、卵泡优势化以及对颗粒细胞功能的影响。同时对卵泡液中EVs及其携带的miRNA的未来研究进行了展望,为卵泡液中EVs及携带的miRNA功能的研究及应用提供了思路和方向。     

细胞囊泡的研究进展

阐述了细胞囊泡出芽、运输、融合的分子机制,并就最新进展进行了综述．以对这个领域有一初步认识。     

一种同时分离培养大鼠肝细胞及肝枯否细胞技术的建立

目的:建立简便、经济的同步分离培养肝细胞及kupffer细胞的方法.方法:采用肝脏原位灌洗结合离体胶原酶灌注消化的方法获得总细胞悬液,差速离心分离肝细胞及肝非实质细胞,经多次低速离心可分离肝细胞,经percoll密度梯度离心以及选择性贴壁法得到纯化的kupffer细胞.台盼蓝染色鉴定细胞活力.使用倒置相差显微镜、HE染色、PAS染色及白蛋白免疫组织化学染色对培养肝细胞的形态及功能进行检测.使用光学显微镜、荧光显微镜及CD68免疫荧光染色鉴定分离的kupffer细胞.结果:体外成功的同步分离培养了肝细胞及kupffer细胞,肝细胞产率为1.37± 0.53× 108/大鼠,kupffer得率为3.45± 0.41×106/g肝脏.细胞存活率及纯度都可达90％.肝细胞培养24h后呈典型肝细胞形态,7天后仍具有糖原合成和白蛋白合成能力.贴壁后的kupffer细胞呈典型的星型或三角形,且其标志分子CD68免疫荧光染色阳性.结论:应用改良的原位灌注方法可以很好的同时分离具有活性及功能的肝细胞和kupffer细胞.     

初断乳大鼠视网膜微血管周细胞的分离培养

探讨、优化初断乳大鼠视网膜微血管周细胞(retinal microvascular pericytes,RMPs)的分离、培养方案。分别从15只初断乳大鼠及15只成年大鼠中剜取眼球,采用眼科显微手术器械分离获取视网膜,经碎化、消化、过滤处理,收集视网膜微血管片段,予接种培养。MTT法测绘RMPs生长曲线,通过倒置显微镜观察RMPs形态,免疫荧光法鉴定周细胞标记物。比较2组之间视网膜分离操作时间、完整性、原代细胞数量、周细胞形态和表面标记物表达。结果显示,初断乳大鼠视网膜均成功分离,其中24眼视网膜呈整片分出,6眼视网膜破裂呈碎片状,单个眼球视网膜分离时间为14.3~45.5 s。视网膜分离操作时间及完整性与成年大鼠差异无统计学意义(P0.05),初断乳大鼠原代RMPs细胞产量较成年大鼠高,细胞增殖能力强,细胞形态及表面标记物与成年大鼠一致。研究结果表明,初断乳大鼠视网膜是一种可用于RMPs培养的良好组织原料,该实验成功建立了初断乳大鼠RMPs分离培养体系。     

Ⅰ型囊泡膜谷氨酸转运体在生后发育大鼠三叉神经运动核中的表达

目的观察Ⅰ型囊泡膜谷氨酸转运体在大鼠三叉神经运动核生后发育过程中的表达变化。方法取生后不同发育阶段大鼠脑干,行冰冻切片和Ⅰ型囊泡膜谷氨酸转运体免疫染色,光镜观察。结果刚出生大鼠三叉神经运动核背外侧部即可以观察到Ⅰ型囊泡膜谷氨酸转运体免疫染色阳性结构,随着发育进展,免疫染色阳性逐渐增加,出生7天后Ⅰ型囊泡膜谷氨酸转运体免疫染色模式接近成年水平。结论大鼠三叉神经运动核内Ⅰ型囊泡膜谷氨酸转运体阳性终末在出生后1周内较快地成熟。     

Ⅱ型胶原酶加压灌流提高成年大鼠心肌细胞分离效率

目的探讨效率更高的成熟心肌细胞分离方法。方法采用Ⅱ型胶原酶升主动脉逆行灌流法分离成年大鼠心肌细胞。对照组采用Langendorff装置灌流;实验组在Langendorff装置基础上加压匀速灌流。在倒置显微镜下观察细胞形态,并计算杆状细胞比率及产量;通过台盼蓝染色和局部场剌激评价心肌细胞活性。结果分离即刻,实验组杆状细胞比率显著高于对照组【(90.3±4.4)%νs.(53.4±5.2)%,P<0.01】;实验组活细胞产量也显著高于对照组[(3.6±0.7)×107νs.(1.9±0.6)×107,P<0.01]。复钙过程中,实验组发生自发性收缩的细胞比率及变圆细胞比率均明显低于对照组【(10.4±2.1)%νs.(18.9±4.5)%,P<0.01;(5.3±1.3)%νs.(8.6±1.7)%,P<0.05】。实验组台盼蓝染色阴性的杆状细胞比率和局部场剌激条件下收缩细胞比率明显高于对照组【(95.3±8.2)%νs.(90.6±9.8)%,P<0.05;(92.2±7.6)%νs.(85±6.4)%,P<0.01】。结论Ⅱ型胶原酶加压灌流可获得高产量、高活性的心肌细胞,提高了成熟心肌细胞的分离效率。     

狂犬病疫苗蔗糖密度梯度离心纯化工艺开发

目的:开发冻干人用狂犬病疫苗(鸡胚成纤维细胞)的连续流蔗糖密度梯度离心纯化工艺。方法:分别比较两种不同初始蔗糖浓度和不同上样速度对纯化效果的影响,初步确定纯化工艺;通过多批次实验确定样品收集范围;比较不同浓缩倍数条件下杂质去除和抗原回收情况,确定合适的收获液浓缩比例;比较不同批次样品纯化后的杂质去除率和重复性,判定本纯化工艺的稳定性。结果:选取60%作为初始蔗糖浓度,在上样速度为150～200ml/min时,可以有效地对10倍浓缩的病毒收获液进行纯化;卵清蛋白、牛血清白蛋白和庆大霉素去除率分别达到99%,95%和95%,且工艺具有极好的稳定性。结论:开发的连续流蔗糖密度梯度离心技术可以作为冻干人用狂犬病疫苗(鸡胚成纤维细胞)的产业化纯化工艺。     

Enrichment for Auxotrophic Mutants of Tetrahymena Using Density Gradient Centrifugation*

SYNOPSIS. A protocol based on density differences between starved and fed cells and employing density gradient centrifugation has been devised to facilitate the isolation of auxotrophic mutants of cell lines derived from Tetrahymena thermophila strain B1868. First, a mass phenotype screening procedure was established whereby true auxotrophic mutants and slow-growing wild-type cells such as strain C* could readily be distinguished. Second, simulation experiments were performed in which wild-type cells starved first in non-nutritive buffer, then suspended in a defined medium lacking a single essential amino acid became significantly denser than the same cells when starved, then suspended in a complete defined medium. Finally, using the same protocol, a reconstruction experiment was carried out which resulted in effective separation of wild-type cells from cells of a tyrosine auxotroph. The overall procedure resulted in a 9-fold increase in the relative frequency of auxotrophic cells, while the density gradient centrifugation alone provided a 400-fold enrichment.     

Rapid Isolation of Metabolically Active Mitochondria from Rat Brain and Subregions Using Percoll Density Gradient Centrifugation

Two procedures are described for isolating free (nonsynaptosomal) mitochondria from rat brain. Both procedures employ a discontinuous Percoll gradient and yield well coupled mitochondria which exhibit high rates of respiratory activity and contain little residual contamination by synaptosomes or myelin. The procedures are considerably more rapid than methods described previously for the isolation of brain mitochondria and do not require an ultracentrifuge or swing-out rotor. The first method separates mitochondria by gradient centrifugation from a P2 (crude mitochondrial) fraction and is likely to be widely applicable for studies in which at least 500 mg of tissue are available as starting material. In the second method, the unfractionated homogenate is subjected directly to gradient centrifugation. This method requires the preparation of more gradients (per gram of tissue) than the first method and yields a subcellular fraction with slightly more synaptosomal contamination. However, this second procedure is more rapid, requires less manipulation of the tissue, and is suitable for obtaining mitochondria with well preserved metabolic characteristics from subregions of single rat brains.     

Dextran不连续密度梯度离心法纯化大鼠胰岛

目的评价Dextran不连续密度梯度离心法纯化大鼠胰岛的效果。方法采用V型胶原酶分离出大鼠胰岛,并应用Dextran不连续密度梯度离心法纯化胰岛。在体视镜下计数双硫腙染色的胰岛并测量染色胰岛的直径。放免法测定胰岛素含量。AO-PI双染色确定胰岛的活力。结果平均每只成年Wistar大鼠的胰腺可分离950±24个胰岛,经Dextran纯化后平均每只成年Wistar大鼠的胰腺可获得784±10个胰岛,纯度可达到90%以上。结论采用Dextran不连续密度梯度纯化得到的Wistar大鼠胰岛结构完整、功能良好。     

Differential Centrifugation in Culture and Differentiation of Rat Neural Stem Cells

(1) The study of neural stem cells (NSC) has attracted much attention in recent years because of their therapeutic potential. However, the problem in culture and differentiation of NSC was how to obtain single cell suspension that preserves the function of NSC, and remove the debris caused by mechanical dissociation. In the present study, we try to find a simple and effective way to address the problem, i.e. differential centrifugation. (2) After a gentle mechanical dissociation using Pasteur pipette, the suspension was first centrifuged at 100 g for 5 min, and then recentrifuged at 400 g for 6 min. Finally, the two deposits were resuspended and seeded into culture flask respectively. The suspension from the second deposit was allowed for further culture and differentiation. Immunofluorescence technique was used to identify neural stem cell, neuron, astrocyte, and oligodendrocyte. (3) After the second differential centrifugation, single cell suspension was obtained with 2–3 cell clusters, and the cells not only grew to form neurospheres, but also differentiated into neurons, astrocytes, and oligodendrocytes. (4) Differential centrifugation is a simple and effective way to obtain single cell suspension, which will help make large-scale production of neurodifferentiated cells more effective.     

出芽短梗霉膨大细胞分选及产糖分析

本研究运用Percoll密度梯度离心的方法对出芽短梗霉Aureobasidium pullulans的两种细胞形态进行分选,并对两种形态的细胞进行多糖产量的分析。通过对转速、分选时间、Percoll分离液浓度的优化,确定了两种细胞形态分选效果最佳的条件是Percoll分离液浓度为60%、转速为5 000r/min、离心时间为30min。经过光学显微镜和透射电子显微镜观察发现上层为酵母状细胞（YL）、下层为膨大细胞（SC）,并发现膨大细胞外有明显的薄膜包被,且产大量多糖。也为今后在相应状态下研究出芽短梗霉膨大细胞的其他代谢机理提供了可行的方法,满足后续研究的需要。     

Separation of Non-filamentous Micro-organisms from Soil by Density Gradient Centrifugation in Percoll

Soil suspensions were homogenized, and desorbed non-filamentous micro-organisms were concentrated in a minimum volume of buffer by low speed centrifugation. The cells were separated from inanimate material by flotation at the interface between the buffer and a silica sol/polyvinyl pyrrolidone density gradient medium (Percoll). Cell suspensions were removed from the interface and fractionated according to density by high speed centrifugation on discriminating density gradients in Percoll.
Preliminary experiments indicated that most non-filamentous soil micro-organisms had densities in the range 1.081–1.123 g%sol;ml while Rhizobium isolated from crushed root nodules on Percoll was split into two bands of densities 1.081–1.110 and 1.041–1.073 g/ml. The lighter cells were the more pleomorphic.
The efficiency of extraction of cells from soil was governed by the extent of their desorption from inanimate particles. As rigorous desorption procedures damage cells, extraction efficiencies were low; 10–20% of cells counted microscopically in soil were recovered from density gradients. Electron microscopy of soil micro-organisms isolated by this method showed an unusual range of surface ornamentations on cell-like structures of bacterial dimensions.     

CsCl Density Gradient Centrifugation Studies of Intact Bacterial Cells

Cells of Escherichia coli have been successfully banded in CsCl density gradients and a portion of the population reclaimed in a viable state. Differentiation between two strains of this organism in a CsCl density gradient has been demonstrated also. Several studies were undertaken to see whether differences could be detected between two samples of cells of the same strain which had been subjected to different conditions. The results were as follows: (a) Introduction of a heavy label (5-bromouracil) into the DNA during a 90 minute period did not produce an observable change in cell density. (b) Removal of a required amino acid from the growth medium of an E. coli auxotroph resulted in an increase in both the density and heterogeneity of the cells. (c) Exposure of cells to 27 kr of gamma radiation, followed by a period during which portions of both DNA and RNA were lost, yielded two distinct bands, one at the normal position in the gradient and the other shifted to a lighter region.     

Separation of Particulate Phytochrome and Plasma Membranes of Maize Coleoptiles by Density Gradient Centrifugation

Phytochrome, cross linked in situ to its receptor by glutaraldehydefixation, and radioactively-labelled membrane material, obtainedby lactoperoxidase-catalysed ¹²⁵I-treatment of maize coleoptilescould be separated from each other according to sedimentationvelocity and by sucrose density gradient centrifugation. Bindingof iodine to membrane material in maize coleoptiles increasedseveral-fold on the addition of reaction-specific enzymes, i.e.lactoperoxidase and glucose oxidase. Mitochondria were considerednot labelled because mito-chondrial purification reduced ¹²⁵Iincorporation. Membrane material containing incorporated iodineappeared quite heterogenous and sedimented to an equilibriumdensity position close to, but slightly lighter than that ofthe mitochondria; participate phytochrome banded at a heavierposition. Results obtained therefore suggest that the receptorfor phytochrome may not be on the plasma membrane as envisagedin recent hypotheses.     

大规模区带离心纯化Vero细胞乙脑疫苗

本文报道一种适合疫苗生产的大规模纯化Ｖｅｒｏ细胞乙脑疫苗的方法。原疫苗经适当浓缩和去除ＤＮＡ处理后,用不连续蔗糖梯度（３６％和６０％）。３２６００ｇ,速率区带离心４ｈ。纯化后疫苗的效力比中国参考疫苗高出６倍以上,补体结合抗原比中国参考疫苗高４～８倍。总蛋白含量低于３０μｇ／ｍＬ,牛血清含量降至０．５μｇ／ｍＬ以下,细胞残余ＤＮＡ低于１００ｐｇ／０．５ｍＬ。用此法连续制备三批纯化疫苗,其纯度和效力均高于日本鼠脑纯化疫苗。此法对于制备其它纯化Ｖｅｒｏ细胞疫苗也具有一定的参考意义。