骨形成发生蛋白对人骨髓间充质干细胞的影响

目的探讨骨形成发生蛋白(BMP)对人骨髓间充质干细胞的影响及调节作用.方法使用含BMP-7基因的PTracer-CMV载体感染人骨髓间充质干细胞(h MSCs),并设未转染组和空载体组,免疫组化法检测BMP-7蛋白表达,MTT法检测细胞增殖能力,流式细胞术检测细胞周期,湿化学法检测碱性磷酸酶合成情况.结果培养48 h后,BMP-7转染组h MSCs增殖速度明显高于未转染组和空载体组,差异具有统计学意义(P0.05);未转染组与空载体组h MSCs增殖速度之间比较差异无统计学意义(P0.05).BMP-7转染组各时间点G_0/G_1期细胞比例均明显低于未转染组和空载体组;S期、G_2/M期的细胞比例均明显高于未转染组和空载体组,上述差异具有统计学意义(P0.05);各时间点未转染组与空载体组G_0/G_1期、S期、G_2/M期细胞比例间差异均无统计学意义(P0.05).BMP-7转染组h MSCs细胞碱性磷酸酶含量明显高于未转染组、空载体组,差异具有统计学意义(P0.05).结论 BMP-7可促进h MSCs体外增殖和向成骨细胞分化,可能与促进细胞由G_1期进入S期、DNA合成增加、提升DNA合成的后期细胞数量有关.     

一种游仆虫无性生殖周期中大核及其DNA含量的变化

应用光学显微镜、显微光度计和定量细胞化学技术研究了一种游仆虫无性生殖周期中大核及其 DNA 含量的变化特征,获得如下结果:(1)游仆虫细胞周期分成 G_1期、S 期和 D 期,各个时期在整个细胞周期中所占的时间速率分别为36%、59%和5%。(2)游仆虫细胞周期中,G_1期大核迅速增大,但此时大核无 DNA 合成现象,DNA 分布密度随大核增大而降低;S 期大核前期增大,此后变小,大核 DNA 含量连续增加,大核 DNA 分布密度也随着增高。8期结束后,大核 DNA 含量相当于 G_1大核的两倍;D期大核又显著增大,DNA 分布密度稍高于 G_1期大核的水平。(3)由游仆虫孚尔根反应大核的形态,大核 DNA 三维吸收图像和显微光度术定量分析数据表明,游仆虫大核 DNA 含量的变化仅发生在 S 期,S 期大核中 DNA 合成与大核改组带相联系。     

离心淘洗技术在药物细胞毒性研究中的应用

应用离心淘洗技术同步分离体外培养小鼠红白血病细胞,得到同步化的G_1期,S期和G_2M期细胞。经药物处理后进行细胞集落形成实验,发现钙拮抗刺异博定对各时相细胞集落形成率影响很小。而抗癌药物平阳霉素对各时相细胞都有较强毒性,特别是对G_2M期细胞,杀伤作用特别强。     

一种游仆虫无性生殖周期中大核及其 DNA 含量的变化

应用光学显微镜、显微光度计和定量细胞化学技术研究了一种游仆虫无性生殖周期中大核及其DNA含量的变化特征,获得如下结果:(1)游仆虫细胞周期分成G_1期、S期和D期,各个时期在整个细胞周期中所占的时间速率分别为36%、59%和5%。(2)游仆虫细胞周期中,G_1期大核迅速增大,但此时大核无DNA合成现象,DNA分布密度随大核增大而降低;S期大核前期增大,此后变小,大核DNA含量连续增加,大核DNA分布密度也随着增高。S期结束后,大核DNA含量相当于G_1大核的两倍;D期大核又显著增大,DNA分布密度稍高于G_1期大核的水平。(3)由游仆虫孚尔根反应大核的形态,大核DNA三维吸收图像和显微光度术定量分析数据表明,游仆虫大核DNA含量的变化仅发生在S期,S期大核中DNA合成与大核改组带相联系。     

taxol对V79—8系细胞微核化机理的探讨

用多种方法探讨了 v_(79-8)系细胞在 taxol 作用下微核化的时期和机理.早熟染色体凝集(PCC)法发现此种 G_1 及 G_2 期缺陷的细胞株,在一定的生长条件下有相当数量的 G_1 和 G_2 细胞存在.taxol 作用后,首先是有丝分裂指数(I_m)的增加,然后随 I_m 的下降细胞微核化增加.秋水仙酰胺阻断的 M 期细胞,在释放的同时加入 taxol,细胞微核化速率增加迅速.微管的间接免疫荧光法显示,在 taxol 作用下,微管集合成不规则的束.微核化细胞可以继续合成 DNA.每个微核中含有可被抗着丝点抗体(ACA)血清荧光染色的着丝点.     

重组hFGF-7腺病毒对角质形成细胞的生物学效应

目的:研究重组腺病毒导入人成纤维细胞生长因子7(hFGF-7)对人皮肤角质形成细胞(HaCat)的生物学效应.方法:通过倍比稀释和感染实验,测定重组腺病毒rAd-hFGF-7的滴度;流式细胞术检测重组腺病毒感染HaCat细胞的感染效率;MTT法检测rAd-hFGF-7对HaCat细胞增殖的影响;重组腺病毒对HaCat细胞周期的影响通过流式细胞术进行检测.结果:高滴度重组腺病毒可高效感染HaCat细胞,其促细胞增殖作用随着重组腺病毒MOI值的增加而增强,当MOI值为50时,感染细胞进入S期和G_2期的细胞比率显著增加.结论:重组腺病毒rAd-hFGF-7可促进HaCat细胞的增殖,改变HaCat细胞周期.     

丁酸钠对M期同步及非同步的HeLa细胞早G_1期阻断的可逆性

本文采用早熟染色体凝集(PCC)和~3H-TdR显微自显影技术,观察丁酸钠对同步及非同步的HeLa细胞早G_1期阻断的可逆性及其可逆的进程。实验发现丁酸钠对HeLa细胞早G_1期阻断完全是可逆的,且这种可逆是在洗去丁酸钠后立即产生,丁酸钠对HeLa细胞早G_1期阻断的时间点发生在细胞进入S期前的9.5h;发现早G_1期阻断后的恢复必须再经过中、晚G_1期然后才能进入S期.     

低氧对食管癌ECa9706细胞生长及顺铂敏感性的影响

着重探讨低氧对食管癌ECa9706细胞生长及顺铂敏感性的影响.ECa9706细胞分别培养于顺铂干预的常氧和低氧(1%O2)环境下,倒置显微镜观察细胞形态,MTT法检测细胞活力,流式细胞法检测细胞凋亡和细胞周期.结果显示:与常氧环境下培养相比,低氧下细胞形态变长、变大,细胞自第7d后生长缓慢.常氧下顺铂作用细胞48h的IC50为(1.180±0.056)μM,低氧下为(2.675±0.063)μM.培养48h后,常氧对照组及常氧高浓度顺铂组(1.18μM)阻滞细胞周期于G1期,低氧下各组细胞周期阻滞于S期.低氧导致顺铂诱导的细胞凋亡率较常氧下降低.表明:低氧环境影响了细胞的形态、增殖及细胞周期,低氧下细胞对顺铂的细胞毒作用敏感性降低.     

EC109细胞株细胞周期DNA代谢和形态改变的研究

人食管癌细胞株 EC109以显微分光光度计测定癌细胞核 DNA 含量,以放射自显影测定细胞核摄取~3H-TdR 和扫描电镜检查细胞表面的超微结构.大多数 EC109瘤细胞间期细胞核的 DNA 含量增加,主要干系细胞 DNA 含量为2 C,属 G_1期,也可见低于四倍体(3 C),高于四倍体(>4 C).甚至多倍体(>8 C)的瘤细胞.~3H-TdR 加入培养基10分钟后,许多瘤细胞核有银粒出现(S 期细胞).大量细胞进入细胞周期反映其恶性程度高.扫描电镜可见 EC109在细胞周期不同阶段其细胞大小形状和微绒毛也不同.本文结果说明用细胞核 DAN 含量测定,~3H-TdR 掺入和细胞表面超微结构可测定肿瘤周期.也可用这些方法判定瘤细胞的恶性程度.     

齐墩果酸联合放疗对HepG2细胞周期和凋亡作用的体外研究

目的体外观察齐墩果酸(oleanolic acid,OA)联合放疗对肝癌HepG2细胞周期和凋亡的影响,探讨OA的放射增敏作用及其机制.方法对数生长期HepG2细胞分为对照组、单纯给药组(OA组)、单纯照射组(IR组)、照射与药物联合作用组(IR+OA组).MTT法检测细胞活力,Hoechst33258染色观察细胞凋亡形态学变化,流式细胞仪检测细胞周期和凋亡,Western blotting检测细胞周期相关蛋白表达.结果 OA显著增加射线的杀伤作用,联合组细胞的活力明显下降,凋亡率增高,细胞周期蛋白CyclinB1和Cdk1的降低,p-Cdk1表达量上升更明显,细胞G2/M期阻滞明显高于对照组(P0.05).结论 OA显著增加射线对肝癌细胞的杀伤作用,其机制可能与CyclinB1-Cdk1复合物的表达量减少和活性抑制、诱导细胞阻滞G2/M期有关.     

Chfr defines a mitotic stress checkpoint that delays entry into metaphase

Chemicals that target microtubules induce mitotic stress by affecting several processes that occur during mitosis. These processes include separation of the centrosomes in prophase, alignment of the chromosomes on the spindle in metaphase and sister-chromatid separation in anaphase. Many human cancers are sensitive to mitotic stress. This sensitivity is being exploited for therapy and implies checkpoint defects. The known mitotic checkpoint genes, which prevent entry into anaphase when the chromosomes are not properly aligned on the mitotic spindle, are, however, rarely inactivated in human cancer. Here we describe the chfr gene, which is inactivated owing to lack of expression or by mutation in four out of eight human cancer cell lines examined. Normal primary cells and tumour cell lines that express wild-type chfr exhibited delayed entry into metaphase when centrosome separation was inhibited by mitotic stress. In contrast, the tumour cell lines that had lost chfr function entered metaphase without delay. Ectopic expression of wild-type chfr restored the cell cycle delay and increased the ability of the cells to survive mitotic stress. Thus, chfr defines a checkpoint that delays entry into metaphase in response to mitotic stress.     

敬钊缨毛蛛蛛毒对人肝癌细胞BEL-7402细胞增殖、细胞周期和C-myc蛋白的影响

通过MTT方法可知敬钊缨毛蛛蛛毒对BEL 7402细胞增殖有较强的抑制作用(p<0.05),时效和量效关系良好.IC50为18mg/L.敬钊缨毛蛛蛛毒可以抑制BEL 7402细胞DNA的合成.流式细胞仪检测发现经过蛛毒作用的BEL 7402细胞凋亡率增加,细胞周期阻滞在G0/G1期.WesternBlot方法进一步检测到蛛毒作用BEL 7402细胞72h后,C myc蛋白表达减弱.实验结果表明,敬钊缨毛蛛蛛毒可以抑制人肝癌细胞BEL 7402的增殖和DNA的合成,其药理作用机理可能除了诱导凋亡外主要是使BEL 7402细胞周期相关蛋白C myc表达减弱,导致细胞周期的变化.     

A role for the nuclear envelope in controlling DNA replication within the cell cycle

In eukaryotes the entire genome is replicated precisely once in each cell cycle. No DNA is re-replicated until passage through mitosis into the next S-phase. We have used a cell-free DNA replication system from Xenopus eggs to determine which mitotic changes permit DNA to re-replicate. The system efficiently replicates sperm chromatin, but no DNA is re-replicated in a single incubation. This letter shows that nuclei replicated in vitro are unable to re-replicate in fresh replication extract until they have passed through mitosis. However, the only mitotic change which is required to permit re-replication is nuclear envelope permeabilization. This suggests a simple model for the control of DNA replication in the cell cycle, whereby an essential replication factor is unable to cross the nuclear envelope but can only gain access to DNA when the nuclear envelope breaks down at mitosis.     

Calcineurin is required to release Xenopus egg extracts from meiotic M phase

Fertilization induces a transient increase in cytoplasmic Ca2+ concentration in animal eggs that releases them from cell cycle arrest in the second meiotic metaphase. In frog eggs, Ca2+ activates Ca2+/calmodulin-activated kinase, which inactivates cytostatic factor, allowing the anaphase-promoting factor to turn on and ubiquitinate cyclins and securin, which returns the cell cycle to interphase. Here we show that the calcium-activated protein phosphatase calcineurin is also important in this process. Calcineurin is transiently activated after adding Ca2+ to egg extracts, and inhibitors of calcineurin such as cyclosporin A (ref. 8) delay the destruction of cyclins, the global dephosphorylation of M-phase-specific phosphoproteins and the re-formation of a fully functional nuclear envelope. We found that a second wave of phosphatase activity directed at mitotic phosphoproteins appears after the spike of calcineurin activity. This activity disappeared the next time the extract entered M phase and reappeared at the end of mitosis. We surmise that inhibition of this second phosphatase activity is important in allowing cells to enter mitosis, and, conversely, that its activation is required for a timely return to interphase. Calcineurin is required to break the deep cell cycle arrest imposed by the Mos-MAP (mitogen-activated protein) kinase pathway, and we show that Fizzy/Cdc20, a key regulator of the anaphase-promoting factor, is an excellent substrate for this phosphatase.     

Movement and segregation of kinetochores experimentally detached from mammalian chromosomes

The kinetochore is a specialized structure at the centromere of eukaryotic chromosomes that attaches chromosomes to the mitotic spindle. Recently, several lines of evidence have suggested that kinetochores may have more than a passive role in the movement of chromosomes during mitosis and meiosis. Kinetochores seem to attract and 'capture' microtubules that grow from the spindle poles and microtubules may lengthen or shorten by the addition or subtraction of tubulin subunits at their kinetochore-associated ends. An attractive hypothesis is that kinetochores function as 'self-contained engines running on a microtubule track'. Here, we show that kinetochores can be experimentally detached from chromosomes when caffeine is applied to Chinese hamster ovary cells that are arrested in the G1/S phase of the cell cycle. The detached kinetochore fragments can still interact with spindle microtubules and complete all the mitotic movements in the absence of other chromosomal components. As these cells enter mitosis before DNA synthesis is completed, chromosome replication need not be a prerequisite for the pairing, alignment and segregation of kinetochores.     

Identification of a novel resident centrosomal protein

One human autoimmune serum was identified to react with centrosomes by immunofluorescence. We applied the affinity purification of membrane-bound antibody technique and demonstrated that the antibodies present in this antiserum reacted with a 31/29 ku centrosomal antigen. Immunofluorescence showed that this antigen is located at centrosome in a cell-cycle independent manner, and thereby it belongs to the family of centrosomal residents. We then uti- lized this autoimmune serum and antibodies against centrin and gamma-tubulin to investigate changes of centrosome cycle kinetics during premature chromosome condensation (PCC) artificially induced in V79-8 cells. We show here that centrosomal proteins continue to express when cells are synchronized at G1/S boundary and S phase by Hydroxyurea (HU). During this time, the addition of caffeine causes cells with unreplicated genome to go into mitosis, and induces the separation of the replicated centrosomes. These results suggest that the coordination of DNA synthesis and centrosome replication in the normal cell cycle can be uncoupled. Cells ensure that centrosome duplicates once, and only once during each DNA synthesis cycle through the tight and subtle coordination of cell cycle engine molecules, and thereby the assembly of bipolar spindle and the accurate transmission of genetic information.     

Evidence that a free-running oscillator drives G1 events in the budding yeast cell cycle.

In yeast and somatic cells, mechanisms ensure cell-cycle events are initiated only when preceding events have been completed. In contrast, interruption of specific cell-cycle processes in early embryonic cells of many organisms does not affect the timing of subsequent events, indicating that cell-cycle events are triggered by a free-running cell-cycle oscillator. Here we present evidence for an independent cell-cycle oscillator in the budding yeast Saccharomyces cerevisiae. We observed periodic activation of events normally restricted to the G1 phase of the cell cycle, in cells lacking mitotic cyclin-dependent kinase activities that are essential for cell-cycle progression. As in embryonic cells, G1 events cycled on schedule, in the absence of S phase or mitosis, with a period similar to the cell-cycle time of wild-type cells. Oscillations of similar periodicity were observed in cells responding to mating pheromone in the absence of G1 cyclin (Cln)- and mitotic cyclin (Clb)-associated kinase activity, indicating that the oscillator may function independently of cyclin-dependent kinase dynamics. We also show that Clb-associated kinase activity is essential for ensuring dependencies by preventing the initiation of new G1 events when cell-cycle progression is delayed.     

Ser10磷酸化的组蛋白H3和微管蛋白在小麦根尖细胞有丝分裂过程中的动态分布

利用间接免疫萎光标记技术研究Ser10磷酸化的组蛋白H3和微管蛋白在小麦根尖细胞中有丝分裂过程中的动态分布情况.结果显示在小麦根尖细胞有丝分裂过程中Ser10磷酸化的组蛋白H3的出现和消失与染色体的凝集和解凝集的过程存在时空上的相关性,在有丝分裂的过程中这种蛋白在着经线粒上的定位有有助于染色体向两极移动.研究结果还表明,在有丝分裂过程中,微管蛋白发生了重组,成束的垂直排列在赤道板的两侧,协助细胞有丝分裂过程的顺利完成.     

The reversibility of mitotic exit in vertebrate cells

A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with a proteasome inhibitor or by expression of non-degradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. However, if after mitotic exit, the Cdk1 inhibitor is washed free from cells in which cyclin B degradation is blocked, the cells can revert back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent degradation of cyclin B provides directionality for the M phase to G1 transition.