大鼠肝癌细胞(CBRH-7919)的甲胎蛋白合成与细胞周期时相的关系

大鼠肝癌细胞(CBRH-7919)是一甲胎蛋白阳性的肝癌细胞系,适用为研究甲胎蛋白基因表达调控的体外模型。本文用免疫荧光法和免疫沉淀法在非同步的和同步的细胞样品中检测了甲胎蛋白在不同周期时相细胞内的合成情况。从免疫荧光反应结合~3H-TdR脉冲标记放射自显影和鉴别不同时相染色方法对比分析,说明该系肝癌细胞在晚G_1期荧光反应很弱,进入早S期胞质荧光明显增强,至晚S期胞质荧光又有减弱。免疫沉淀法检测同步的晚G_1、早S和晚S期细胞的结果,与细胞学所得结果一致。甲胎蛋白量是早S期高于晚S期,而S期又明显地高于晚G_1期。早S期是该系肝癌细胞合成甲胎蛋白的高峰时相。甲胎蛋白合成在不同时相细胞之间有明显差异,提示控制细胞周期进程有可能对甲胎蛋白基因的表达进行调控。     

人胃癌不同周期细胞膜表面ConA受体的分布与侧向运动

本文研究了人胃低分化粘液性腺癌细胞MGC 80-3不同周期时相中ConA受体的分布与侧向运动。MGc 80-3细胞经同步化培养,用F-ConA标记。被标记细胞中G_1、S和G_2期呈不连续的分布,但它们之间又存在显著的差异。M期呈较均匀的强荧光分布(与其它时相细胞比较)。荧光漂白恢复方法测定ConA受体复合物侧向运动表明:各个周期时相之间不仅运动方式不同,而且运动速率也有显著差异。M期与G_1期主要表现出扩散型运动;而S期与G_2期表现为流动型运动。G_1期的扩散系数大干M期的;S期的流动速率大于G_2期的。但可动分子百分比以G_2期最高。这些结果表明了ConA受体的动力学性质。它受到细胞周期的调节。     

细胞周期蛋白和细胞周期

细胞周期是当前细胞生物学和分子生物学的热门研究课题之一。细胞生物学家、分子生物学家和遗传学家们运用了不同的材料对细胞周期G_1→S、S→G_2、G_2→M和M→G_1等过渡的调控问题进行了研究,其中以周期蛋白和p34~(cdc2)相互作用的研究最为引人注目。在G_2→M过渡期,p34~(cdc2)和周期蛋白B结合,经历一系列的磷酸化和去磷酸化过程,形成有活性的MPF,实现G_2→M期的过渡,在M期中/后期,周期蛋白B降解,致使MPF失活,细胞离开M期;在G_1→S期过渡时,p34~(cdc2)和G_1周期蛋白结合,引起DNA的复制,使细胞进入S期。此外还发现不少cdc 2的类似物在细胞周期各过渡期也有作用。因此,不同的周期蛋白和不同的cdc 2产物,参与细胞周期的方式不同。细胞周期的调控是多途径的和多层次的,这已成为进一步揭示细胞周期调控活动所必需研究的问题。     

Taxol诱导CHO细胞微核化的动态分析

本实验观察到10μM Taxol对CHO细胞的G_1→S→G_2→M期的进程不产生影响,但使CHO的有丝分裂中期大大延长,不能形成纺锤体,凝集的染色体散在于胞质中,不形成中期板。经Taxol处理的CHO细胞的TM要比正常CHO细胞TM长13倍,不能完成胞质分裂,形成带有3—15个微核的细胞,最小的微核只含有1个染色体的DNA含量,微核化细胞形成率可高达96%左右。微核化细胞可进入下一个细胞周期,形成更多微核的细胞。用1×10~(-4)M二硝基苯酚(DNP)不影响微核化细胞的形成,加进10微克/毫升环己亚胺后7小时使微核化细胞形成速率减慢。     

大蒜油不同给药途径对癌细胞周期移行过程的影响

本研究采用流式细胞光度术(FCM)分析癌灶局部及肌注大蒜油(GO)后,S180腹水癌细胞周期移行过程的变化。实验结果表明癌灶局部注射 GO 后,组方图上高倍体 DNA 峰值减少,癌细胞由 G_1期向 S 期运行受阻,S 期细胞显著减少,G_1期与 Go 期细胞积累。肌注 Go 对癌细胞周期影响远不如癌灶局部给药明显。Go 与 G_1期癌细胞积累的原因可能与 GO 对癌细胞周期活动选择性的抑制,或特异性杀伤 S 期或 G_2+M 期癌细胞有关。     

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。     

P15INK4B高表达对人黑色素瘤细胞G1/S进程的阻滞及与MAPK信号相关性之初探

利用TdR-N_2O同步法分别获得P15高表达的MLIK6和表达空载体的MLC2的M期细胞和G_1期细胞,~3H-TdR掺入结果显示,与对照组细胞MLC2相比,实验组细胞MLIK6从G_1期进入S期时间延长2h,并且掺入强度明显减弱,DNA合成被抑制。进一步观察了P15~(INK4B)对G_1/S相关调控蛋白的影响,在M期细胞释放8h(晚G_1期细胞)后,与对照组MLC2细胞相比,实验组MLIK6细胞中CyclinD1,CyclinE,Cdk4,C-Myc蛋白水平均降低。相反,P27~(KIP1)的表达却上升。同时探讨了MAPK信号在P15~(INK4B)阻抑A375细胞G_1/S转换中的作用与相关性,结果显示晚G_1期的MLIK6细胞中ERK1,ERK2水平变化不大,而具有活性的P-ERK1和P-ERK2均表现出下降。上述实验表明,P15~(INK4B)可能通过作用于G_1期相关的周期调节蛋白和抑制ERK1和ERK2活性,阻滞G_1/S转换与抑制DNA合成。     

肝癌细胞与内皮细胞的粘附力学特性研究

采用细胞同步技术和微管吸吮技术,从细胞周期的角度研究不同细胞周期肝癌细胞(hepatocellularcarcinoma cells,HCC)与脐静脉内皮细胞(HUVEC)的粘附力学特性。结果表明,未同步化肝癌细胞各周期时相的细胞百分比为:G_0/G_1期,53.51％;G_2/M期,11.01％;S期,35.48％。采用胸腺嘧啶脱氧核苷、秋水仙碱顺序阻断和胸腺嘧啶脱氧核苷双阻断后释放培养的方法可分别获得G_1期和S期的肝癌细胞,其平均同步率分别为69.02％和96.50％。G_1期肝癌细胞与人脐静脉内皮细胞的粘附力比S期相应值明显降低(P<0.01),与未同步化肝癌细胞组比较也得到同样结果,而S期与未同步化肝癌细胞组的粘附力值无明显差别。肝癌细胞与脐静脉内皮细胞的粘附力随着粘附时间的变化而变化,在30～60min内迅速增长,60min之后维持在较稳定的水平,即300×10~(10)N左右。提示:在肝癌细胞与内皮细胞的粘附过程中,S期细胞可能起的作用更大;肝癌细胞和内皮细胞上粘附分子表达呈现时间效应,从而体现出粘附和去粘附的行为特征。     

铝酞菁:人癌细胞的吸收及光敏杀伤的研究

本文比较了光敏剂铝酞菁(AISPC)及血卟啉衍生物(HPD)在人癌细胞的吸收及光敏杀伤中的差异.探讨了两光敏剂在细胞中的分布、摄入量,血清的影响、及细胞对两光敏剂的吸收动力学过程,结果显示,除了类似的特征外,细胞对AISPC的吸收速度慢于HPD.光敏杀伤的结果表明,在红光照射下,两光敏剂对细胞有相近的杀伤效率;在室内自然光下,AISPC对细胞的损伤明显低于HPD,显示出其较低的光毒付作用,具有适合临床应用的优点.     

竹红菌甲素对体外培养细胞光敏作用的实验研究

本文用免疫荧光细胞化学、流式荧光分析等方法研究了光敏剂H_A对培养的HeLa细胞的光敏化作用。结果表明,H_A光敏化作用使细胞形态发生变化,细胞表面微绒毛丧失,细胞增殖受到抑制,上述变化随光照剂量增加而加剧,其中对肿瘤细胞的抑制作用较正常二倍体细胞更大,细胞群体中G_1期细胞下降,S期细胞增多。H_A的光敏化作用还使胞质微管变稀疏,微管中心的亮荧光近乎消失。以上实验说明,H_A的光敏作用对培养细胞的作用引起细胞膜、细胞骨架的损伤,使细胞周期部分阻断于S期,从而抑制了细胞的增殖。     

主动脉平滑肌细胞增殖周期中磷酸酪氨酸蛋白的研究

以人工合成的磷酸酪氨酸牛血清白蛋白为抗原免疫家兔,得到抗TPK 天然磷酸化底物(P-tyr-Pr)的特异性抗体。P-tyr-Pr ELISA测定结果显示:ASMC 胞浆和核内颗粒部分P-tyr-Pr 的最高含量均出现于G_1晚期(9 h),且核内P-tyr-Pr 的含量远高于胞浆。免疫组织化学显示:ASMC 中P-tyr-Pr 的总量也以G_1晚期为最高,在G_1晚期ASMC 中,P-tyr-Pr 主要分布于细胞核及其周围的胞浆中。     

FIP200对FN诱导的人类呼吸道上皮细胞增殖的影响

目的研究 FIP200(FAK-family interacting protein of 200 kDa)对细胞外基质成份诱导的呼吸道上皮细胞增殖和细胞周期演进的影响及其作用机制.方法通过纤连接蛋白(FN)诱导培养呼吸道上皮细胞增殖,以FIP200反义寡核苷酸(ODN)经脂质体转染细胞;利用四唑盐(MTT)比色实验研究呼吸道上皮细胞增殖情况;采用流式细胞术分析呼吸道上皮细胞细胞周期各期分布情况;以Western blot检测FIP200和FAK蛋白表达水平;以免疫共沉淀检测FIP200和FAK结合情况和FAK磷酸化程度.结果 FN诱导的呼吸道上皮细胞MTT吸光度明显增强,G1期细胞数量明显减少,S期和G2期细胞数量明显增多,同时FAK表达水平及其酪氨酸磷酸化程度显著增高,FIP200表达有降低趋势,FAK与FIP200的结合程度显著减低;与40mg/L FN组相比,经脂质体转染了反义FIP200寡核苷酸的气道上皮细胞MTT吸光度值显著增高,G1期细胞数明显减少,S期和G2期细胞数显著增多,FIP200表达水平显著降低,FAK表达水平无明显变化,与FIP200结合的FAK显著降低, FAK酪氨酸磷酸化程度明显增高.结论内源性FIP200和FAK的结合抑制FAK的活化,从而抑制呼吸道上皮细胞增殖和细胞周期演进,内源性FIP200作为FAK的抑制剂而存在;FN能够促使FAK和FIP200结合的解离而活化FAK,从而促进细胞增殖.     

巨细胞病毒感染致MRC-5细胞周期及Cyclin E表达的变化

本文研究了巨细胞病毒感染人二倍体细胞MRC-5后诱导产生高水平Cyclin E,Cyclin E／cdk2激酶活性增加和导致细胞周期阻滞。应用流式细胞仪分析表明10PFU／cell的病毒量感染MRC-5细胞72h后,29％细胞位于S期,69％细胞位于G2/M期,只有2％细胞位于G1／G0期。应用双抗夹心ELISA法检测,感染病毒20h后,MRC-5细胞中Cyclin E含量比对照细胞高出8倍。感染细胞中Cyclin E／cdk2激酶活性基本上与Cyclin E含量相关联。     

EFFECTS OF PHYTOCHROME AND BLUE-NEAR ULTRAVIOLET LIGHT-ABSORBING PIGMENT ON DURATION OF COMPONENT PHASES OF THE CELL CYCLE IN ADIANTUM GAMETOPHYTES

Single-celled protonemata of the fern Adiantum capillus-veneris, kept under continuous red light, grew with a very low rate of cell division, and the cell cycle was arrested in the early G₁ phase. Cell division was induced by transferring the protonemata to the dark after various light treatments, and the duration of component phases in the cell cycle was determined by a continuous-labelling technique with 3H-thymidine. Blue light irradiation greatly reduced the duration of the G₁ phase but did not affect that of other phases. The greater the fluence of blue light, the shorter was the duration of G₁ phase was observed. In contrast, a brief exposure of red-light-grown protonemata to far-red light given immediately before the dark incubation showed no effect on the duration of G₁ S and M phases but significantly extended that of the G₂ phase. The effect of far-red light on the G₂ phase was reversed by red light, and the effects of red and far-red light were repeatedly reversible. The progression in the M phase was shown by means of a time-lapse video system to be not at all influenced by any pre-irradiation described above.     

乙酰胆碱及其拮抗剂对人SK-N-SH细胞增殖及mAchRl表达的影响

用CCK-8比色法和流式细胞术,检测乙酰胆碱(acetylcholine,Ach)及拮抗剂阿托品(at-ropine,Atro)对SK-N-SH细胞增殖活性和周期分布的调节作用;进一步用荧光定量PCR、免疫印迹和流式细胞间接免疫荧光技术,分析SK-N-SH细胞毒蕈碱受体亚型Ⅰ型(mAchR1)和c-fos的表达差异。结果表明,1mmol/LAch对SK-N-SH细胞有明显促增殖作用,而1mmol/LAtro阻滞细胞从S期向G_2/M期移行;1mmol/LAch与1mmol/LAtro均反馈调节mAchR1的蛋白水平,但mAchR1mRNA的表达不受影响;1mmol/LAch显著上调c-fosmRNA和Fos蛋白的表达,但这种作用可被Atro逆转。提示胆碱类受体参与配基对肿瘤细胞的促增殖作用。     

Glutathione is recruited into the nucleus in early phases of cell proliferation

We have studied the possible correlation between nuclear glutathione distribution and the progression of the cell cycle. The former was studied by confocal microscopy using 5-chloromethyl fluorescein diacetate and the latter by flow cytometry and protein expression of Id2 and p107. In proliferating cells, when 41% of them were in the S+G(2)/M phase of the cell cycle GSH was located mainly in the nucleus. When cells reached confluence (G(0)/G(1)) GSH was localized in the cytoplasm with a perinuclear distribution. The nucleus/cytoplasm fluorescence ratio for GSH reached a maximal mean value of 4.2 +/- 0.8 at 6 h after cell plating. A ratio higher than 2 was maintained during exponential cell growth. In the G(0)/G(1) phase of the cell cycle, the nucleus/cytoplasm GSH ratio decreased to values close to 1. We report here that cells concentrate GSH in the nucleus in the early phases of cell growth, when most of the cells are in an active division phase, and that GSH redistributes uniformly between the nucleus and the cytoplasm when cells reach confluence.     

Differentiation of mitotic melanoma cells from G2 cells and their isolation by use of 5-bromo-2'-deoxyuridine and propidium iodide

This report describes a method by which mitotic cells were isolated from nonsynchronized Cloudman melanoma cells that had been pulse labeled with 5-bromo-2'-deoxyuridine (BrdUrd) and double-stained with a fluoresceinated monoclonal antibody to BrdUrd and with propidium iodide (PI). In initial experiments, melanoma cells were first pulse labeled with BrdUrd, treated with prostaglandin E1 (PGE1 10 micrograms/m1) or vehicle (0.1% ethanol) for up to 24 hours, then stained with anti-BrdUrd and PI. PGE1-treated cells monitored at 3-hour intervals were observed to migrate from S phase to G2 phase, then, enigmatically, back into the late S phase region of the distribution. In other experiments, cells treated with PGE1 were pulse labeled with BrdUrd at the end of the treatment period and harvested. In these experiments, there was a small, discrete subpopulation of cells within the late S phase region of the DNA distribution that was negative for anti-BrdUrd. This subpopulation of cells was sorted and examined by light microscopy. We observed that 95% of these BrdUrd-negative "S phase" cells were mitotic cells. Since mitotic cells and G2 cells have equivalent amounts of DNA, the reduced red fluorescence exhibited by these cells may be due to a greater sensitivity to denaturation, which has been described for DNA of mitotic cells, and would account for the phenomenon of cells appearing to move "backwards" in the cell cycle. This report indicates that although the BrdUrd/PI method can further define the cell cycle into four compartments, it can also lead to over-estimation of S phase cells in kinetic studies because of contaminating mitotic cells.