济南假单胞制剂PJV对大鼠肝大颗粒淋巴细胞数量、酶活性及自然杀伤活性的影响

本文通过细胞分离、细胞化学染色、电镜观察及~3H-TdR释放试验体外检测细胞毒活性等方法,研究了在生物反应调节剂—济南假单胞制剂PJV作用下,大鼠肝大颗粒淋巴细胞(LGL)和枯否细胞(KC)的数量、形态及其免疫生物特性等变化。结果表明,PJV的应用可引起肝内LGL数量增长达4倍,其体外杀伤肿瘤细胞活性增长约2倍,细胞内ACP活性明显提高。肝KC数量及活性也有明显增加。本文还讨论了肝LGL与KC的相互关系。     

重度子痫前期患者外周血NK 细胞功能的研究*

目的:探讨重度子痫前期患者外周血中NK细胞数量、杀伤功能以及相关细胞因子的变化。方法:选择重度子痫前期患者25例(研究组)以及正常妊娠妇女25例(对照组),流式细胞术测定外周血中NK细胞数量,细胞毒实验测定NK细胞的杀伤活性,ELISA法测定血清IFN-γ及IL-2的浓度。结果:研究组患者外周血中NK细胞的数量以及杀伤活性显著高于对照组;研究组外周血中IFN-γ、IL-2的浓度也显著高于对照组。结论:重度子痫前期患者外周血NK细胞数量增多,杀伤活性增强,血清中NK细胞相关细胞因子也增加。这些改变可能参与重度子痫前期的发病机制。     

免疫荧光磁粒分离诱导CD34＋细胞转化为DC的实验研究

目的：建立一种磁荧光分离细胞的方法将CD34＋细胞转化为DC。方法：人脐带血单个核细胞,经免疫免疫荧光标记、外磁场分离CD34＋细胞,分别加入GM—CSF、IL-4和不同浓度黄芪多糖,诱导12—14天检测DC细胞数量和功能。结果：随黄芪多糖浓度的升高,DC细胞的百分率越大。结论：黄芪多糖可作为一种优良的诱导促进剂,联合使用GM-CSF和IL-4可将CD34＋细胞转化成为DC。     

小鼠血管损伤后巨噬细胞与平滑肌细胞的相互作用

目的研究血管损伤后病变形成过程中的巨噬细胞与平滑肌细胞的相互作用。方法C57BL/6（6～8周龄）小鼠24只,右侧股动脉植入透明塑料微导管,制作小鼠血管损伤模型,术后给予特异性抗体AFS98及APB5,分别阻断巨噬细胞和平滑肌细胞增殖的信息传导通路。给药2周后采集股动脉组织,用免疫组织化学的方法对血管病变进行分析。结果小鼠股动脉血管损伤2周后,病变部位聚集了大量的巨噬细胞、平滑肌细胞。给予阻断巨噬细胞增殖的信息传导通路的特异性抗体AFS98后,病变部位的巨噬细胞数量显著减少,平滑肌细胞数量反而增多。相反,给予抑制平滑肌细胞增殖的抗体APB5后,病变局部平滑肌细胞数量减少,而巨噬细胞数量急剧增加。结论小鼠股动脉血管损伤后,构成病变的细胞主要为巨噬细胞与平滑肌细胞。这两种细胞在分化成终末成熟细胞的过程中,存在着相互拮抗的作用。     

胰腺β细胞的发育与代偿

传统观念认为,出生时就已拥有了终生的胰腺β细胞（由它产生胰岛素）。然而,现有证据表明,β细胞群是动态的,通过β细胞功能和β细胞群大小数量的变化来维持血糖浓度局限于一个很狭窄的生理范围内。β细胞群大小数量的增加或减小,可能与β细胞数量（增殖或凋亡）和单个细胞体积（肥大或萎缩）的变化有关。当机体不能精确调控β细胞群的大小数量时,就会发生糖尿病。因此,了解β细胞的发育分化、干细胞起源和代偿机制对防治糖尿病具有重要意义。本文简要介绍这方面取得的进展。     

三种底栖淡水鱼类皮肤黏液细胞分布与数量比较

采用常规石蜡切片以及AB-PAS染色方法研究了葛氏鲈塘鳢（Perccottus glenii）、黄颡鱼（Pelteobagrus fulvidraco）以及泥鳅（Misgurnus anguillicaudatus）3种底栖淡水鱼类的皮肤黏液细胞类型以及分布,计数10个视野下（视野面积为43.5 μm × 32.6 μm）3种鱼类头部、背部、腹以及尾部皮肤的黏液细胞数量,并用单因素方差分析（ANOVA）比较鱼体4种黏液细胞数量差异。结果表明：（1）3种鱼类的主要黏液细胞不同,葛氏鲈塘鳢鱼体黏液细胞中Ⅲ型细胞居多,较Ⅰ型细胞多61.5%,较Ⅱ型细胞多85.8%,较Ⅳ型细胞多85.7%;在黄颡鱼体表,Ⅰ型黏液细胞分布数量最多,较Ⅱ型细胞多9.9%,较Ⅲ型细胞多15.1%,较Ⅳ型细胞多53.5%;而在泥鳅体表以Ⅱ型细胞数量最为丰富,较Ⅰ型细胞多88.3%,较Ⅲ型细胞多33.1%,较Ⅳ型细胞多83.5%。（2）对于葛氏鲈塘鳢,黏液细胞集中分布在头部,比背部黏液细胞数量多15.4%,比腹部黏液细胞数量多出38.0%,比尾部黏液细胞数量多56.7%;黄颡鱼以背部黏液细胞数量为多,比头部黏液细胞数量多42.5%,比腹部黏液细胞数量多46.6%,比尾部黏液细胞数量多51.4%;泥鳅也在背部具有丰富的黏液细胞,比头部黏液细胞数量多49.9%,比腹部黏液细胞数量50.6%。（3）3种鱼类之间的黏液细胞总数不同,泥鳅体表的平均黏液细胞数量最多,相较于葛氏鲈塘鳢多38.9%,较黄颡鱼多39.1%。研究结果表明,不同鱼类的鱼体表面黏液细胞种类不同,可能与其生活环境和鱼体本身的特性有关。     

不同胚龄北京鸭视网膜节细胞层细胞大小、数量及密度

采用视网膜铺片Nissl染色法和Scion Image图像分析法,研究北京鸭胚胎期11（E11）、14、117、20、23和26日龄视网膜节细胞层（Ganglion cell layer,GCL）细胞大小、数量及密度分布变化。结果表明：E11-E14,GCL细胞小,均呈圆形,E17开始出现大细胞;E11至E26视网膜中央区（CA）细胞大小增长1．97倍,而颞侧周边区（TP）增长3．1倍,相邻胚龄间细胞大小差异显著。GCL细胞总数在E17达到最大值2．03×10^6个,随后细胞数量显著下降。视网膜中央-周边密度梯度在E11出现。CA细胞密度在E17达到最大值2．54×10^4个/mm^2,随后密度下降;周边区细胞密度随胚龄增长而持续下降,且TP细胞密度下降最显著。结果提示,北京鸭胚胎发育过程中,GCL细胞大小、数量及密度均发生显著变化,而E17是其中的一个重要转折点[动物学报54（6）：1082—1088,2008]。     

秦岭木姜子油细胞的分布和结构研究

利用组织透明法和石蜡制片法研究秦岭木姜子各器官内油细胞的结果表明：其根、茎、叶和果实内部有油细胞分布。但它们在大小、数量上存在差异：根的皮层和茎的次生木质部射线中的油细胞体积最小,直径１６￣２５μｍ,且数量较少;果实果肉中的油细胞体积最大,直径约７０μｍ,数量也最多：根的次生韧皮部薄壁组织、茎的皮层和髓以及叶肉组织中的油细胞大小和数量则介于前两者之间,直径约４０μｍ。油细胞多数为单个散在,呈球形或     

颌下腺导管细胞与胶原海绵细胞相容性的实验研究

为探讨胶原海绵对颌下腺 (submandibulargland ,SMG)导管细胞的细胞相容性 ,采用HE染色光镜观察及免疫组化观察SMG导管细胞接种于胶原海绵后 ,细胞的生长情况。光镜下可见接种后第 1d细胞数量较少 ,分散于胶原海绵支架中间 ,第 7d细胞数量明显增加 ,免疫组织化学染色抗IV型胶原抗体染色呈阳性 ,说明细胞与支架材料之间已经有细胞外基质产生。胶原海绵具有良好的细胞相容性 ,是一种理想的支架材料。与胶原海绵复合培养 ,颌下腺导管细胞仍可保持良好的增殖能力。     

人慢性胃炎与神经内分泌G、D细胞关系的研究

探讨神经内分泌G、D细胞与慢性胃炎的关系,用免疫细胞化学方法对52例慢性胃炎及9例对照者进行胃窦粘膜内G、D细胞密度计数。结果显示慢性胃炎病人的G、D细胞数均低于对照组,特别是萎缩性胃炎G、D细胞显著减少,同时还发现不同组别、不同程度的萎缩性胃炎,其G细胞的数量均大于D细胞,G／D细胞的比值在中、重度性胃炎与其它组相比有显著性差异（P＜0.01）。资料还显示G、D细胞的计数不仅可以判断胃窦粘膜的萎缩程度,而且可作为观察临床疗效的一项重要指标。     

Dynamics of single cell property distributions in Chinese hamster ovary cell cultures monitored and controlled with automated flow cytometry

Two important variables that are often not measured online in Chinese hamster ovary (CHO) cell cultures are cell number concentration and culture viability. We have developed an automated flow cytometry system that measured the cell number concentration, single cell viability based on propidium iodide (PI) exclusion, and single cell light scattering from bioreactor samples every 30 min. The bioreactor was monitored during batch growth, and then the cell number concentration was controlled at a set point during cytostat operation. NH₄Cl was added during steady state operation in cytostat mode to monitor the transient cell population response to adverse growth conditions. The automated measurements correlated well to cell concentration and viability determined manually using a hemacytometer. The described system provides a method to study mammalian cell culture physiology and dynamics in great detail. It presents a new method for the monitoring and control of animal cell culture.     

桉树树干维管形成层和次生韧皮部热致细胞坏死的定量试验

桉树树干维管形成层和次生韧皮部热致细胞坏死的定量试验 桉树(Eucalyptus)树干暴露在森林火灾辐射热中会杀死形成层细胞及其内嵌的再生分生组织,阻止树木萌枝和恢复。目前尚无组织水平的方法来量化热处理对桉树形成层细胞活力的影响。本研究的目 标包括：(1)采用并验证四氮唑还原法检测桉树细胞活力;(2)应用该方法确定斜叶桉(Eucalyptus oblique)形成层细胞活力的阈值水平,进而确定临界温度。采用四氮唑还原法量化该桉树韧皮部-形成层细胞活力。 从斜叶桉成树上切下带有形成层和韧皮部的圆形树皮切片,将质量为1-30 mg不等的样品在20–85°C 的温 度处理中放置1分钟,并在室温条件下在0.8%的2,3,5-氯化三苯基四氮唑(TTC)中保存20–22小时以检测 细胞活力。用乙醇冷萃取得到1,3,5-三苯基四唑甲臜(TPF),在485 nm处测定吸光度。结果表明,TTC还原 法准确地量化了组织切片(包括维管形成层)中细胞活力随温度升高而下降的情况,并确定60°C 为桉树物种形成层-韧皮部细胞的临界温度。细胞活力按[TPF 处理温度]/[TPF 20°C]计算,在20-85°C之间下降90%。细胞活力结果证实,在50-70°C的温度区间,经过1分钟体外组织加热,桉树的组织坏死显著 增加。TTC 方法显示细胞活力随温度升高而下降,这与温度处理和中性红染色处理后独立获得的活细胞计 数一致。     

Automated monitoring of cell concentration and viability using an image analysis system

In order to automate measurements of cell concentration and viability in a suspended animal cell culture, we have developed anin situ microscopic image analysis system with an effective cell recognition algorithm. With a small amount of sample, this system can measure the cell density rapidly and aseptically. In addition, it can measure a cell size histogram including cell debris small particle distribution. These small particles have been found to be related to the viability of the mouse-mouse hybridoma STK1 cell line. By using cell debris small particle density as an indicator of cell viability, the developed system provides non-destructive viability monitoring without trypan blue staining.     

定点整合人Bcl-2基因的CHO/dhfr-细胞株的建立

构建重组载体质粒pMCEfrt—Bcl-2,利用FIp—In^TM定点重组系统,在CHO—dhfr^-细胞内定点整合人Bcl-2基因,通过Western印迹检测重组细胞Bcl-2蛋白的表达。通过流式细胞仪和DNA Ladder检测在高NH4C1条件下细胞的凋亡情况;用台盼蓝染色检测在无血清IMDM培养基中细胞的活细胞数目和活细胞比例。结果获得了稳定表达Bcl-2基因的细胞株CHO—Bcl-2,该细胞株能高水平表达Bcl-2蛋白。在无血清培养过程中,CHO—Bcl-2细胞比对照细胞保持高约15％的活细胞比例,细胞总数高25％。CHO-Bcl-2在高NH4^＋（50mmol／L）培养条件下具有较低的凋亡水平。建立了能够高表达Bcl-2基因并具有一定的抗凋亡能力的重组CHO／dhfr^-细胞株。     

Effects of different detachment procedures on viability,nitroxide reduction kinetics and plasma membrane heterogeneity of V‐79 cells

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G₁). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.     

Cell hybridization by electrofusion on filters

Electric field pulses induce permeabilization and associated fusogenicity in cell membranes. Electrofusion of cells is usually performed in two steps: the first is the creation of close intercellular contacts; the second is an application of electric pulses that induces membrane fusion. Very large cell contacts can be obtained by a filter aspiration method. A cell monolayer is created by controlled suction on biocompatible filter. No spontaneous fusion results. Just after filtration, electrofusion is obtained by field pulses applied parallel to the filter. Cell viability is not strongly affected and cells recover their spherical shape in the minute time range after filtration. The electrical parameters, the cell density, and the flow rate control fusion. Fusion is obtained with cells of different origins with very different adhesion properties. Hybrid cells are easily formed. This approach appears to be a very efficient method for cell hybridization with an easy-to-use protocol.     

马头山羊成纤维细胞系的建立与生物学特性分析

采用组织块贴壁培养法对马头山羊耳缘组织进行培养, 成功构建了马头山羊耳缘组织成纤维细胞系, 并对其形态学、生长动力学、细胞活力测定、中期染色体、微生物污染等特性进行了研究。结果表明: 培养细胞形态为典型的成纤维细胞, 细胞群体倍增时间(PDT)约为36 h。细胞冻存复苏后的活率为96.7%, 传代后生长状况与冻存前一致。细胞中期染色体二倍体(2n=60)占主体约为96%。微生物检测细菌、真菌、病毒支原体检测结果为阴性。细胞系各项指标均达到美国典型培养中心(ATCC)标准。此细胞库的建立在细胞水平上对马头山羊的遗传资源进行了保存, 也为今后的生物学研究以及体细胞克隆保种等研究提供了理想的实验材料。     

微囊化K562细胞生长周期及代谢特性的研究

以K562细胞为模型,分别进行微囊化和游离培养,运用流式细胞术考察两种培养体系下细胞周期和生长代谢变化;建立数学模型,模拟了两种培养体系下细胞的生长活性和代谢特性。实验发现：微囊化培养过程中的K562细胞处于DNA合成期（S期）的百分含量显著高于游离培养,并且细胞保持较高的增殖活性。模型计算表明,所建模型动力学参数能够很好地描述微囊化和游离两种培养体系下细胞的代谢情况;对细胞活性的理论计算表明,微囊化的细胞具有较高的增殖和代谢活性,同时细胞能够较长时间保持此活性;模型参数表明,两种培养体系下,葡萄糖对细胞生长的影响无显著差别 (k^Free_L≈k^APA_L),乳酸对游离培养细胞的生长具有明显抑制作用,但对微囊化培养细胞抑制作用较小(kFreeL>≈kAPAL)。     

Voltammetric behavior of the MCF-7 cell cytoplasm and the effect of taxol on voltammetric response

The aims of this study were to find the electroactive species in the human breast cancer (MCF-7) cell cytoplasm causing a voltammetric response of the cells and to establish a simple and rapid measurement method to obtain strong and direct electrochemical responses objectively reflecting the cell viability. Ultrasonication was carried out for the electrochemical detection. The presence of guanine and xanthine in the MCF-7 cell eluent secreted by the living cells and in the MCF-7 cell cytoplasm was verified by HPLC assay with a DAD system and chemometric method. The concentrations of guanine and xanthine in the MCF-7 cell cytoplasm and the voltammetric response of the MCF-7 cell cytoplasm had higher levels than those of intact cell suspensions. Additionally, taxol caused a decrease of the voltammetric response of the cytoplasm and concentrations of xanthine and guanine in the cytoplasm. Therefore, the origin of the voltammetric response of the MCF-7 cytoplasm was driven by the alteration of the levels of xanthine and guanine, which were related to the cell viability. Thus, the voltammetric response of the ultrasonicated MCF-7 cell suspension could be used to monitor the MCF-7 cell growth and to evaluate the effectiveness of antitumor drugs on tumor suppression.     

Co-culture of glomerular epithelial cells and mesangial cells on collagen-gauze-fiber gel

A novel collagen-gauze-fiber gel was created as a scaffold for the co-culture of renal glomerular epithelial cells and mesangial cells at its opposite sides. This gauze-fiber-gel provides a mimic environment like that of renal glomeruli in vivo. The cell morphology, cell growth and cell viability were investigated and the results showed that this novel scaffold maintains cell growth and cell viability without changing cell morphology for more than 3 weeks. Interestingly, glomerular epithelial cells co-cultured with mesangial cells on the gauze-fiber gel resulted in the polarity formation which usually appears on the normal epithelial cells existing at glomerular basement membrane in vivo, but seldom appears on the epithelial cells when cultured in vitro.