用生物素-亲和素ELISA法检测人胚肺二倍体细胞培养的甲型肝炎病毒抗原

利用生物素-亲和素生物放大系统的原理,建立了新的检测细胞培养的甲型肝炎病毒抗原的方法。用生物素标记的抗甲肝IgG,与免疫偶联于固相的甲肝抗原作用,然后用亲和素-生物素辣根酶复合物(ABPC)与生物素化的IgG偶合,邻苯二胺(OPD)显色。该法在检测甲肝抗原中获得满意结果,灵敏度比普通ELISA法提高约10倍,非特异性显著降低。该法可进一步发展成为一种高特异性的,灵敏的甲肝临床诊断和流行病学调查的技术。     

用人胚肺二倍体细胞(2BS和SL7)分离甲型肝炎病毒

用我国建株的两株人胚肺二倍体细胞(2BS和SL7),以35℃为培养温度,直接从急性期甲型肝炎(简称甲肝)患者粪便中分离到4株病毒。该病毒在细胞培养上符合甲肝病毒的增殖特性,经免疫荧光阻断、免疫电镜及中和试验等特异性鉴定,证实为甲肝病毒。原始分离时的比较研究还显示,2BS株人二倍体细胞支持甲肝病毒增殖的能力似较SL7细胞佳。该4株病毒现已在2BS细胞稳定传代,感染滴度较高,被用于减毒活疫苗的育种研究。     

CHANGES IN CELL PROLIFERATION KINETICS OCCURRING DURING THE LIFE HISTORY OF MONOLAYER CULTURES OF A MOUSE TUMOUR CELL LINE

Following inoculation of monolayer cultures of EMT6 mouse tumour cells at 10⁵ cells, a short lag is followed by 3 days of exponential growth with a population doubling time of 12 hr. A plateau cell number is reached between days 4 and 5 and is maintained for at least 8 days. During exponential growth, the pulse ³H-TdR labelling index is 55–60%, all cells are in cycle, and the median cycle time is 11–12 hr. For the first 3 days of plateau phase, the labelling index is about 25 % and there is considerable cell loss. The cell cycle is 32–40 hr, and S-phase is very long. Later in plateau phase, the labelling index falls to <2 % and there is little cell loss. The changes in kinetics occurring in EMT6 cultures are discussed with reference to reported changes occurring in other cell lines.     

人肺鳞癌和腺癌组织中c-erbB-2癌基因蛋白表达与细胞增殖及预后的关系

用S－P免疫组织化学方法,用c－erbB－2多克隆抗体及PCNA单克隆抗体对50例肺鳞癌和30例腺癌组织石蜡包埋标本进行检测。结果显示：c－erbB－2染色总阳性率为46．25％,鳞癌阳性率为40％,腺癌阳性率为56．67％,两者之间无显著性差异。伴有淋巴结转移的肺癌组织中c－erbB－2阳性表达率较高（66．67％）,并且与肺癌预后有关。PCNA表达阳性级别与患者的术后生存期呈负相关（P＜0．05）,与c－erbB－2表达阳性级别呈正相关（P＜0．05）。本研究提示,用免疫组织化学方法检查,c－erbB－2蛋白和PCNA共同表达可作为临床判断肺鳞癌和腺癌预后的较好指标之一。     

QUANTIFICATION OF ENTRY INTO AND EXIT FROM THE CELL CYCLE IN HUMAN LYMPHOCYTE CULTURES

A mathematical model is presented for the analysis of transition between cycling and non-cycling compartments by cells responding to a growth stimulus. the cellular age distribution as a function of time is derived from sequential [³H]thymidine pulse labeling indices. Rates of entry into and exit from the cycling compartment are determined on the basis of labeling indices obtained after instantaneous and long duration [³H]thymidine pulses. Analysis of an experiment involving sequential measurements over the whole lifespan of a human lymphocyte culture stimulated by phytohemagglutinin is presented as an example of the application of this method.     

SUBCULTIVATIONS, SPLITS, DOUBLINGS, AND GENERATIONS IN CULTURES OF HUMAN DIPLOID FIBROBLASTS

Mathematical expressions are provided relating subcultivations to population doublings and population doublings to generations. Account is made for the increasing number of cells which do not attach upon subcultivation as well as the increasing number of non-dividers. The expressions reconcile published differences in longevity and reveal that the in vitro lifetime of human diploid cells measured in cell generations is far greater than previously suspected.     

ANALYSIS OF CELL PROLIFERATION DURING EARLY EMBRYOGENESIS

Cell proliferation was examined during early embryogenesis of the newt ( Triturus pyrrhogaster ) by various methods. After the two-cell stage, at 23°C, the blastomere (cell) number per whole embryo increased logarithmically until the mid-blastula stage (for about 19 hr) and the rate of increase slowed down in and after the late blastula stage. On the other hand, the synchronous cleavage of the blastomeres at the animal pole continued for 18 hr until the twelfth cleavage (mid-blastula) and the transition from synchronous to asynchronous division occurred abruptly at and after the thirteenth cell division (late blastula). The study also showed that the presumptive neuro-ectoderm consisted mainly of cells of the fifteenth generation (G-15) at the onset of gastrulation (pigment stage).
The present study suggested that the number of ectodermal cells of the early gastrula (stage 12a) nearly doubled during gastrulation at the presumptive neuro-ectoderm. This means that most of the ectodermal cells are in G-16 at the end of gastrulation. On the other hand, both mitotic activity and the rate of cell increase gradually diminished during gastrulation in the ectoderms of both the presumptive neural and epidermal regions, and there are evidently significant differences in both activities between the neuro-ectoderm and the epidermal ectoderm after stage 13b: the epidermal ectoderm showed greater decrease in the rate of both mitotic activity and cell proliferation than the neuro-ectoderm.
These facts suggested that, whether the ectodermal cells will differentiate into neural cells or epidermal cells is determined during G-15 or G-16 in normal primary induction.     

细胞松弛素B对人胚肺二倍体细胞SL_7和肿瘤细胞CNE、LTEP-78增殖周期的不同影响

CB(1.5微克/毫升)处理正常二倍体细胞SL_7和人肺鳞癌细胞LTEP-78及鼻咽癌细胞CNE,以~3H-TdR掺入和DNA含量变化为指标,观察十天内,核复制动态。SL_7细胞~3h-TdR掺入进行性下降至0,细胞周期被迫阻断(非时相特异性阻留),一部分细胞成为双核。肿瘤细胞LTEP-78和CNE,~3H-TdR掺入受一定程度抑制,但细胞周期移行可以持续,结果,产生一部分多核细胞和染??色体多倍化。CB引起的微丝束解聚与~3H-TdR掺入下降之间,未见到相关性。     

急性低氧下去甲肾上腺素对大鼠淋巴细胞转化的调节作用

本研究以模拟高原低氧方法,观察低氧作用于大鼠细胞免疫功能以及去甲肾上腺素对免疫作用的调节机制。实验结果：与对照相比,７ｋｍ急性低氧２４ｈ淋巴细胞转化下降４１％（Ｐ〈０．０１）;５ｋｍ低氧暴露时间为７ｄ,２０ｄ时,低氧抑制淋巴细胞转化,分别下降３４％和６０％（Ｐ〈０．０１）,侧脑室注入５ｎｍｏｌ／Ｌ ＮＥ,淋巴细胞转化比对照下降２９％（Ｐ〈０．０１）,７ｋｍ１０ｈ低氧暴露时,侧脑室注入酚妥拉明２５μ     

THE HUMAN CIRCADIAN CLOCK AND AGING

The suprachiasmatic nucleus (SCN) of the hypothalamus is implicatedin the timing of a wide variety of circadian processes. Since the environmentallight-dark cycle is the main zeitgeber for many of the rhythms, photic informationmay have a synchronizing effect on the endogenous clock of the SCN by inducingperiodic changes in the biological activity of certain groups of neurons.By studying the brains obtained at autopsy of human subjects, marked diurnaloscillations were observed in the neuropeptide content of the SCN. Vasopressin,for example, one of the most abundant peptides in the human SCN, exhibiteda diurnal rhythm, with low values at night and peak values during the earlymorning. However, with advancing age, these diurnal fluctuations deteriorated,leading to a disrupted cycle with a reduced amplitude in elderly people. Thesefindings suggest that the synthesis of some peptides in the human SCN exhibitsan endogenous circadian rhythmicity, and that the temporal organization ofthese rhythms becomes progressively disturbed in senescence. (ChronobiologyInternational, 17(3), 245–259, 2000)     

CELL PROLIFERATION AND SPECIALIZATION DURING ENDOCHONDRAL OSTEOGENESIS IN YOUNG RATS

Endochondral osteogenesis was studied autoradiographically in ribs and tibiae of 32 Long-Evans rats injected with 1 µc/gm H³-thymidine at 6 days of age and sacrificed at intervals between 1 hour and 2 weeks later. Proliferation and specialization of bone cells were studied by analyses of (a) the percentage of mitoses which were labeled, (b) the percentage of labeled nuclei in bone cells, and (c) grain counts. The following conclusions were derived: The various types of bone cells represent different functional states of the same cell. Cell division is usually restricted to cells in the morphologically unspecialized "osteoprogenitor" state. Specialized bone cells arise by modulation of osteoprogenitor cells. The average cell generation time is shortest in the metaphysis, longest in the periosteum, and intermediate in the endosteum. The average duration of DNA synthesis is relatively constant (about 8 hours). With increasing length of generation time there is a slight increase in G₂ + mitosis, but the major change is a lengthening of G₁. After dividing, cells in the osteoprogenitor state may remain within the progenitor pool or undergo modulation of cell type, specializing as osteoblasts or becoming incorporated in osteoclasts.     

前体、诱导子及抑制剂对细胞培养生产紫杉醇的调节作用

研究了前体、诱导子和抑制剂对中国红豆杉细胞培养生产紫杉醇的影响。结果表明 ,它们之间的协同作用对提高紫杉醇的产量有显著影响。其中向培养基中加入 30mg/L 3 甲基 2 丁烯 1 醇 ,2mmol/L苯甲酸钠 ,10mg/L氯化氯胆碱 (CCC) ,10 0 μmol/L茉莉酸甲酯 (MJ) ,0 1mmol/L丝氨酸 (Ser)可以使紫杉醇含量增加 1141 1%。     

钙调素抑制剂——三氟拉嗪对肿瘤细胞增殖和微管组装的影响

本文用钙调素抑制剂——三氟拉嗪处理人胃癌MGC-803细胞,用免疫荧光细胞化学方法,放射免疫法和速流荧光分析等方法研究了钙调素对细胞增殖,环核苷酸代谢及微管组装,有丝分裂等细胞功能的调节作用。实验结果表明,TFP明显地抑制了人胃癌细胞的增殖,这种抑制增殖的作用,具有剂量和时间依赖关系,细胞群体中G_1期细胞增多,S期细胞下降,DNA合成明显地受到抑制。TFP处理的胃癌细胞仅在短时间内(5'-30')cAMP含量升高,cGMP浓度降低,cAMP/ ??cGMP比值比对照组高4.4倍,但此后环核苷酸含量又很快恢复到对照组水平。本实验还观察到TFP处理后的MGC-803细胞胞质铺展,细胞形态的改变与胞质微管的分布有密切联系,实验结果表明TFP加强了人胃癌细胞MTOC对微管的组装能力,使微管分布得到恢复,微管纤维呈放射状延伸到细胞边缘,充满胞浆,使细胞呈现出展平的多边形,趋向于正常上皮细胞形态的变化,本实验结果表明TFP抑制癌细胞增殖及使微管组装加强可能是通过对CaM活性的抑制作用。此结果有助于说明转化细胞内钙调素的变化,可能是与转化细胞增殖失控和胞质微管消退有关。     

用人胚肺二倍体细胞株分离一株甲型肝炎病毒

自甲型肝炎患者粪便中分离到一株甲型肝炎病毒(TZ-84)。病毒不产生细胞病变,胞浆中有颗粒性荧光。细胞中的病毒抗原能被抗甲肝病毒抗体阳性血清所中和。 TZ-84株病毒耐酸,耐乙醚。免疫电镜见有大量直径27～30mm的甲肝病毒颗粒,以空心为主。将其接种细胞后的黑暗期随传代而缩短,病毒滴度则随传代而增加。将细胞粉碎后获得的病毒。ELISA滴度为1:16。用其检测45份人血清中抗HAV-IgM抗体,并与Abbott ELISA药盒比较,符合率为91.1％,敏感度为87.1％。     

KINETIC ANALYSIS OF CELL SIZE AND DNA CONTENT DISTRIBUTIONS DURING TUMOR CELL PROLIFERATION: EHRLICH ASCITES TUMOR STUDY

In order to study the growth dynamics of proliferating and non-proliferating cells utilizing discrete-time state equations, the cell cycle was divided into a finite number of age compartments. In analysing tumor growth, the kinetic parameters associated with a retardation in the growth rate of tumors were characterized by computer simulation in which the simulated results of the growth curve, the growth fraction, and the mean generation time were adjusted to fit the experimental data. The cell age distribution during the period of growth was obtained and by a linear transformation of the state transition matrices, was employed to specify the cell size and DNA content distributions. In an application of the model, the time-course behavior of cell cycle parameters of Ehrlich ascites tumor is illustrated, and the parameters important for the transition of cells in the proliferating compartment to the non-proliferating compartment are discussed, particularly in relation to the G₁-G₀ and G₂-G₀ transitions of non-cycling cells as revealed by the variation of cell size distribution.     

4－甲基组胺和三氟啦嗪对WEHI_3细胞增殖的影响

用体外细胞液体培养法研究组胺Ｈ＿２受体激动剂４－甲基组胺（４－ＭＨ）、Ｈ＿２受体拮抗剂西咪替丁和钙调素拮抗剂三氟啦嗪（ＴＦＰ）对小鼠粒单白血病ＷＥＨＩ＿３细胞增殖的影响。结果表明：４－ＭＨ呈剂量依赖性抑制ＷＥＨＩ＿３细胞增殖。１０￣（－４）ｍｏｌ／Ｌ西咪替丁可阻断这种效应。１０￣（－５）─１０￣（－４）ｍｏｌ／ＬＴＦＰ阻断ＷＥＨＩ＿３细胞增殖,１０￣（－６）ｍｏｌ／ＬＴＦＰ对细胞影响不大,但与高浓度４－ＭＨ合用有协同抑制效应。     

从九连小檗细胞培养物中分离和鉴定药根碱

九连小檗的叶片,在含有2,4-D(1mg/l),KT(0.1mg/l),琼脂(0.8%),蔗糖(2%)的B,培养基上,诱导出愈伤组织,在25±2℃条件下进行固体、液体培养、收集悬浮培养20天的细胞进行化学抽提和分离,得到一种橙黄色结晶,经 HPLC、UV、IR、MS 和 NMR 等分析鉴定,证明为药根碱。经药理试验表现有明显的生理活性。     

Survivin靶向siRNA抑制大肠癌细胞增殖的作用

目的 构建survivin靶向siRNA重组表达载体,研究其对人大肠癌细胞生长的抑制作用。方法构建Stirvivin靶向siRNA并转染结肠癌细胞,通过RT-PCR和Westernblot方法检测survivin的表达,采用MTT法检测siRNA对细胞增殖的抑制作用。结果经酶切鉴定和测序结果证实survivin靶向siRNA重组表达载体构建成功,它对大肠癌细胞survivinmRNA和蛋白的表达抑制率分别为36．33％和44．65％,肿瘤细胞的生长受到明显抑制。结论survivin靶向siRNA构建成功并能显著抑制survivin基因的表达。     

钙调素对细胞周期的调节

RC3细胞是一种用真核表达载体1~(CaM)转染NIH 3T3细胞建成的可调钙凋素(Calmodulin,CaM)高表达细胞模型。通过分子杂交及蛋白免疫印迹方法证实在地塞米松(Dexamethasome,DXM)作用下,RC3细胞可高表达CaM。CaM的过表达使G_1期细胞减少,S期细胞增加;CaM拮抗剂三氟拉嗪(trifluoperazine,TFP)则使G_1期细胞增加,S期细胞减少。高表达CaM使细胞分裂指数提高,G_2期细胞减少,有丝分裂前期细胞增加,M中期细胞比例下降。而TFP处理则使分裂指数下降,G_2期细胞增加,M前期细胞减少,M中期细胞增加。实验结果表明CaM在G_1/S、G_2/M和M中期/M后期3个位点上对细胞周期进行调控;通过加速G_1至S期,G_2至M期和M中期至M后期的进程,使细胞倍增时间缩短,促进细胞增殖。本工作表明,RC3细胞作为CaM表达可调细胞模型,是研究细胞周期调控的有力工具。