保罗样激酶基因沉默体外抑制胶质瘤细胞侵袭与生长

目的观察保罗样激酶1基因(plk1)沉默对胶质瘤细胞株-H4体外生长的抑制作用,探讨plk1基因作为胶质瘤治疗靶点的可行性。方法化学合成小片断干扰RNA(siRNA)抑制plk1基因的表达,Western blot检测plk1蛋白质的表达变化,流式细胞仪检测H4细胞周期分布及凋亡程度的变化,体外侵袭实验检测H4细胞侵袭能力的变化,MTT法检测H4细胞增殖速度的变化。结果经siRNA作用48h后,plk1蛋白质水平明显降低;较多的H4细胞聚集于G2/M期附近(P<0.05);细胞凋亡明显上升(P<0.05);细胞体外侵袭能力下降(P<0.05);增殖速度明显缓于对照组(P< 0.05)。结论靶向plk1的siRNA可在体外抑制胶质瘤细胞H4的侵袭与增殖,plk1有可能成为新的潜在胶质瘤治疗靶点。     

A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension

Kinetochores play an essential role in chromosome segregation by forming dynamic connections with spindle microtubules. Here, we identify a set of 10 copurifying kinetochore proteins from Caenorhabditis elegans, seven of which were previously uncharacterized. Using in vivo assays to monitor chromosome segregation, kinetochore assembly, and the mechanical stability of chromosome-microtubule attachments, we show that this copurifying protein network plays a central role at the kinetochore-microtubule interface. In addition, our analysis suggests that the network is comprised of three groups of proteins that contribute in distinct ways to this interface: KNL proteins act after the assembly of centromeric chromatin to generate the core of the microtubule-binding interface, MIS proteins control the rate and extent of formation of this interface, and NDC proteins are necessary to sustain tension during interactions with spindle microtubules. We also purify a similar set of associated proteins from human cells that includes four novel proteins and has recognizable homologs from each functional class. Thus, this protein network is a conserved constituent of the outer kinetochore, and the functions defined by our analysis in C. elegans are likely to be widely relevant.     

Effect of α-difluoromethylornithine on the polyamine levels and proliferation in two transplantable tumours

Summary The effect of inhibition of polyamine biosynthesis by-difluoromethylornithine (DFMO) on the growth of two murine transplantable tumours was studied. Female CBA mice were implanted with either the sarcoma F (SaF) or an anaplastic mammary carcinoma (CaNT), and 3% DFMO in the drinking water was provided once the tumours were established. Over a 10-day period control SaF tumours increased exponentially from 20 mm³ to over 800 mm³, whereas DFMO-treated SaF reached only 300 mm³. CaNT grew more slowly, requiring 22 days to achieve a similar volume increase, and DFMO was as effective in retarding growth as it had been in SaF. DFMO depleted tumour tissues of putrescine and spermidine, but did not reduce spermine levels. Metaphase arrest experiments with vincristine demonstrated that DFMO could substantially reduce the rates of tumour cell production, but there was no indication the DFMO accelerated the rate of cell loss from the tumours. Despite reduced rates of cell production, labelling studies with bromodeoxyuridine failed to detect differences between control and treated tumours: an increase in transit time through the S-phase was suspected. The number of nuclear organizer regions, detected by the argyrophilia of their associated proteins, was less in DFMO-treated tumours, and within a tumour the degree of silver deposition unequivocally reflected the proliferative heterogeneity. Ultrastructural studies revealed no differences between DFMO-treated and untreated tumours.     

肾母细胞瘤经动脉化疗栓塞术疗效机制探讨

目的： 探讨肾母细胞瘤术前经动脉化疗栓塞的疗效机制。方法: 17例肾母细胞瘤患儿介入治疗后再行肿瘤切除，另 22例行单纯手术切除。对照分析2组肾母细胞瘤样本中细胞和间质的变化，采用原位细胞凋亡(TUNEL 法) 检测瘤细胞凋亡，并通过免疫组化法检测瘤细胞P53、Bcl-2 和Bax蛋白的表达。术后随访2年以上。结果: 介入组与单纯手术组的肿瘤坏死区域平均面积分别为60%、15 %（Uc = 2.84）；肿瘤组织呈X₃、X₄退变者分别为58.8%（10/17）和4.55%（1/22）（2c= 11.4）、间质纤维组织增生程度中、重度变化者分别为64.7%（11/17）和18.2%（4/22）（Uc = 2.72）、淋巴细胞侵润者分别为70.6%（12/17）和18.2%（4/22）（2c=10.9），2组有显著差异（P<0.01）；介入组肿瘤细胞分裂指数的中位数为0.2/10高倍视野，明显低于单纯手术组的1.4/10高倍视野（Uc = 54.50 ，P<0.01）；94.9(37/39)的病例中均有不同数量的阳性凋亡细胞出现。介入组肿瘤细胞凋亡指数的中位数为25.9/10高倍视野，明显高于单纯手术组的12.8/10高倍视野（Uc = 117.00，P<0.05）。P53和Bcl-2蛋白表达与瘤细胞凋亡及分化程度均无相关性（P>0.05），但Bax 蛋白在介入组瘤细胞表达率（80.0%）明显高于单纯手术组（40.0%），差异显著（P<0.05）。介入组2年无瘤生存率为73.3%（11/15），单纯手术组为42.9%（6/14）。结论: 经动脉化疗栓塞治疗能够有效杀伤肿瘤细胞、抑制肿瘤生长，诱导由Bax蛋白介导的肿瘤细胞凋亡。     

Mitotic index and multipolar mitosis in routine histologic sections as prognostic markers of pancreatic cancers: A clinicopathological study

ObjectivesPancreatic cancer is characterized by genomic complexity and chromosomal instability, and atypical mitotic figures are morphological features of this phenotype. In the present study, we determined the frequency and the clinicopathological and prognostic significance of mitotic figures in pancreatic cancers.MethodsWe surveyed the mitotic figures of the normal ductal epithelium, acinar cells, pancreatic intraepithelial neoplasias, and pancreatic cancers on hematoxylin-and-eosin–stained tissue specimens (n = 121).ResultsPancreatic cancer cells showed significantly higher mitotic indices as compared with the ductal cells, acinar cells, and pancreatic intraepithelial neoplasias. Both normal and atypical mitosis were significantly elevated only in pancreatic cancers. In pancreatic cancers, approximately 30% of total mitosis was atypical including multipolar, lag-type, ring and asymmetrical mitosis, and anaphase bridges. The Kaplan–Meier curves in pancreatic cancers showed significant correlations between total mitosis and disease free survival. Furthermore, the cases with multipolar mitosis showed poorer prognosis than those without. Lymph node metastasis and multipolar mitosis were independent prognostic factors for overall survival of patients with pancreatic cancer. In addition, lymph node metastasis and total mitosis were independent factors for disease free survival.ConclusionThese findings suggest that routinely obtained pathological specimens, even small biopsy or cytological specimens, can provide valuable information concerning the prognosis of pancreatic cancers.     

Changes in DNA content,number of nuclei and cellular dimensions of young rat atrial myocytes in response to left coronary artery ligation

Studies of enzymatically isolated myocytes from atria of young male Sprague-Dawley rats at 11 days after left coronary artery ligation show that a major response of atrial myocytes to ventricular infarction is binucleation. In sham-operated animals, 23.2% of left and 15.5% of right atrial myocytes were binucleated, compared to 77.8% of left and 40.5% of right atrial myocytes of infarcted animals. Examination of 150 g and 250 g unoperated control animals indicate that this response is occurring at a time when a small but significant amount of binucleation is also occurring as a normal part of growth. Using a Feulgen-acriflavine-SO₂ method for cytofluorometry, a significant increase in ploidy was seen in left atrial myocytes of infarcted animals over those of sham or control animals. The number of left atrial myocytes in infarcted animals having a ploidy level above 3C was 10.8% above sham values. The mean length of binucleated myocytes of left atrium was significantly greater in infarcted animals (119.8 μm) than in sham-operated animals (97 μm) and the mean length of mononucleated myocytes was greater in infarcted animals (104.1 μm) than in sham-operated animals (77 μm). Thus, cardiac myocytes are capable of a substantial response to a stressful situation by increases in cell length, number of nuclei and ploidy. Study of a model system such as the rat atrium may yield an understanding of the mechanisms involved in the induction of these nuclear changes.     

Early stage epithelial ovarian cancers: A study of morphologic prognostic factors

We intended to reevaluate the morphologic prognostic factors for early-stage ovarian carcinomas. We reviewed 111 patients diagnosed with early-stage ovarian cancer who had undergone primary surgery at Hacettepe Hospital between 1984 and 2001, using diagnostic criteria from the WHO-2003 classification. We applied the Universal grading system suggested by Shimizu/Silverberg and noted FIGO-stage, histotype, tumor size, bilaterality, and endometriosis. These features were compared with each other and survival. The survival analysis was carried out by Kaplan–Meier curves. Of the cases, 52 were reclassified as ‘borderline tumor’ or ‘cystadenoma with borderline foci’ and 59 as ‘invasive carcinoma’. FIGO-stage and mitotic count were significant for survivals of 59 patients with cancer. Mitotic index was also significant for the probability of metastasis. The patients with stage-II cancer had 5.65 times more risk of recurrence than stage-I cancer. The 5-year overall and disease-free survivals rates were 90.6% and 87.5% for stage-I, 54.7% and 39.3% for stage-II, respectively. Universal grade did not reach statistical significance for survivals but it was related to FIGO-stage significantly. In conclusion, FIGO-stage is the most reliable prognosticator. Although prognostic value of universal grade is not significant, mitotic count may provide important prognostic information for early-stage ovarian carcinomas.     

Indicine N-oxide binds to tubulin at a distinct site and inhibits the assembly of microtubules: A mechanism for its cytotoxic activity

Indicine N-oxide, a pyrrolizidine alkaloid present in the plant Heliotropium indicum had shown promising cytotoxic activity in various tumor models. The compound exhibited severe toxicity to hepatocytes and bone marrow cells. The present work was aimed to evaluate the molecular mechanism of the toxicity of indicine N-oxide. We found that indicine N-oxide inhibited the proliferation of various cancer cell lines in a concentration dependent manner with IC₅₀ ranging from 46 to 100 μM. At the half maximal inhibitory concentration it blocked the cell cycle progression at mitosis without significantly altering the organization of the spindle and interphase microtubules. The toxicities of the compound at higher concentrations are attributed to its severe depolymerizing effect on both the interphase and spindle microtubules. Binding studies using purified goat brain tubulin indicated that indicine N-oxide binds to tubulin at a distinct site not shared by colchicine or taxol. It decreased the polymer mass of both purified tubulin and MAP-rich tubulin. It was found to induce cleavage of DNA using pUC18 plasmid. The interactions of indicine N-oxide on DNA were also confirmed by computational analysis; which predicted its binding site at the minor groove of DNA. These studies bring to light that the toxicities of indicine N-oxide were due to its DNA damaging effects and depolymerization of microtubules.     

染色体驱动蛋白KIF4A稳定低表达胃癌细胞系的构建及鉴定

目的 构建稳定低表达染色体驱动蛋白KIF4A的胃癌细胞系,观察KIF4A低表达细胞的有丝分裂期纺锤体中央区的形成.方法 针对人类驱动蛋白KIF4A mRNA的编码区设计特异性siRNA序列,构建KIF4A短发夹RNA（shRNA）表达质粒pGPU-GFP-KIF4A.将质粒转染胃癌细胞SGC-7901,经过G418筛选获得稳定低表达KIF4A的细胞株.使用蛋白免疫印迹方法鉴定细胞株内KIF4A蛋白的敲降效果,并通过细胞免疫荧光染色法观察细胞纺锤体中央区的形成.结果 成功获得了3株不同水平稳定低表达KIF4A的SGC-7901细胞株（SGC-shKIF4A）和稳定表达无意义shRNA的对照细胞（SGC-shNC）;同时,与对照细胞SGC-shNC相比,SGC-shKIF4A细胞中有丝分裂纺锤体中央区延长,并随KIF4A蛋白表达水平的降低而增加.结论 成功构建了不同水平稳定低表达KIF4A蛋白的胃癌细胞SGC-shKIF4A,为进一步研究驱动蛋白分子KIF4A的功能及其在胃癌发生发展过程中的作用奠定基础.