乳腺癌干细胞相关亚群细胞周期分析

目的：流式细胞仪分选乳腺癌细胞系MCF-7的肿瘤干细胞相关SP亚群（side population，SP），并检测SP和非SP细胞周期。方法：MCF-7细胞悬液经Hoeehst33342染色，流式细胞仪分选SP和非SP后，乙醇固定，PI染色，流式细胞仪检测细胞周期。结果：SP细胞在MCF-7细胞中占4％，Vempamil阻断后比例减为0．5％。SP细胞中S／G2／M期细胞最低，仅占7．9％，而非SP细胞增殖期比例相对高，S／G2／M期细胞约占34．2％。未分选细胞中S／G2／M期细胞比例居中，为22．9％。结论：人乳腺癌细胞系MCF-7含SP亚群，比例约为4％，且SP细胞多处于静止期，具有一般干细胞的特性。     

通过改造工程细胞提高重组蛋白类药物产量的研究进展

哺乳动物细胞是表达蛋白类生物产品的理想宿主细胞。但是,表达水平常较低。通过对工程细胞进行改造,如抑制细胞凋亡、对细胞周期进行调控、提高翻译后加工能力、改变细胞新陈代谢的特性、实现基因组热点整合等,可有效地提高重组蛋白类药物的产量,为生物药品的规模化生产奠定基础     

Exploring the anticancer activities of novel bioactive compounds derived from endophytic fungi: mechanisms of action,current challenges and future perspectives

Cancer is the second leading cause of death all around the world. The natural compounds derived from the endophytic flora of fungi are possible solutions to cancer treatment because they are safe for health, cost-effective, biocompatible and have fewer toxicity issues. The active ingredients in endophytic fungi that are responsible for anti-cancer activities are alkaloids, terpenoids, glycosides, saponin, peptides, steroids, phenols, quinones, and flavonoids. This review highlights the anti-cancer activities of entophytic fungus against human papillary thyroid carcinoma (IHH4), human pancreatic (PANC-1), ovarian (OVCAR-3), hepatic (HepG2), lung (A-549), human lymphoma (U937), human skin carcinoma (A431), breast (MCF-7), and Kaposi’s sarcoma. The emerging evidence suggested that bioactive compounds isolated from endophytic fungi showed their anti-cancer activities by revealing the disturbance of the microtubule network caused by increased levels of Bax and Bcl-2 proteins that triggers cell cycle arrest at the G2-M phase, by inhibiting the DNA replication via binding with topoisomerase II, by regulating the activity of extracellular signal-regulated kinase and NF-kB, by evaluating the levels of p21, p27, and cyclins B/D1/E that led to cell death by apoptosis and cell cycle arrest. This review will assist readers in better comprehending bioactive chemicals and the beneficial interaction between the fungal endophytes and medicinal plants.     

PC-SPESⅡ对雄激素非依赖型前列腺癌裸鼠移植瘤G1/S期及凋亡相关蛋白表达的影响

目的研究PC-SPESⅡ影响AIPCa细胞周期进展及凋亡的分子机制。方法80只雄性Balb/c-nu/nu裸小鼠分为2大组,分别接种DU145或PC-3细胞,建立AIPCa移植瘤模型,每组再分成对照、PC-SPESⅡ125、250、500mg/kg4个剂量组,每组10只,接种第2天起每天给药,8周后各组中随机抽取3份标本,Westernblot和SABC免疫组织化学法检测G1/S期调节蛋白P16、P21、P-Rb及凋亡相关蛋白Bax、Bcl-2、Cleaved-PARP表达的变化。结果Western blot:用药后DU145组P16表达显著升高(P=0.001),P21和P-Rb表达无变化(P=0.188和P=0.778);Bax、Cleaved-PARP表达升高(P=0.003和P=0.002),Bcl-2显著下降(P=0.001)。PC-3组P16、P21呈剂量依赖性上升(P<0.0001),P-Rb呈剂量依赖性降低(P=0.005);Bax无显著变化,每天500mg/kg时Bcl-2显著降低(P=0.021),Cleaved-PARP升高(P=0.02)。免疫组化:PC-3组6种蛋白及DU145组P16、P-Rb、Bax与Western blot结果一致。DU145组P21阳性率呈剂量依赖性升高(P=0.001),Bcl-2、Cleaved-PARP表现出更明显的剂量依赖性降低或升高趋势。结论PC-SPESⅡ可以通过调节G1/S阻滞和凋亡相关蛋白表达抑制AIPCa生长。     

A Novel Model of Ultrasonic Fatigue Test in Pure Bending

The very high cycle fatigue (VHCF) failure of in-service components is mainly caused by the vibration of thin-wall elements at a high frequency. In this work, a novel model of ultrasonic fatigue test was developed to test thin-wall material in bending up to VHCF with an accelerated frequency. The theoretical principle and finite element analysis were introduced for designing a sample that resonated at the frequency of 20 kHz in flexural vibration. In the advantage of the second-order flexural vibration, the gauge section of the sample was in the pure bending condition which prevented the intricate stress condition for thin-wall material as in the root of cantilever or the contact point of three points bending. Moreover, combining the constraint and the loading contact in one small section significantly reduced heating that originated from the friction at an ultrasonic frequency. Both strain gauge and deflection angle methods were applied to verify the controlling of stress amplitude. The fractography observation on Ti6Al4V samples indicated that the characterized fracture obtained from the novel model was the same as that from the conventional bending test.     

端粒酶反义寡核苷酸对宫颈癌HeLa细胞端粒酶活性的影响

目的　探讨端粒酶反义寡核苷酸 (ASON)对宫颈癌HeLa细胞体外生长抑制的作用机制。方法　针对端粒酶RNA的ASON作用于HeLa细胞 ,台盼蓝排斥法测定细胞存活力 ,以PCR为基础的TRAP法测定端粒酶的活性 ,流式细胞仪 (FCM)检测细胞凋亡及细胞周期。结果　ASON连续治疗 5d ,HeLa细胞的存活力无明显下降 ;在FuGENE6的介导下 ,ASON对端粒酶活性的抑制与ASON的浓度和治疗时间呈正相关。FCM未检测到特征性的亚G1 峰 ,ASON组中G2 /M期的DNA含量显著增多。结论　针对端粒酶RNA的ASON能抑制HeLa细胞的端粒酶活性 ,并将细胞阻滞在分裂期达到抑制细胞生长的目的     

The MDR1 (P-glycoprotein) and MRP (P-190) transporters do not play a major role in the intrinsic multiple drug resistance of Jurkat T lymphocytes

The response of T cells in relation to the cell cycle has not been extensively studied. We have attempted to address this question using Jurkat T cells treated with cytostatic drugs known to arrest cells at various transition points of their cycle. We tested several concentrations of drugs that act at G1/S (hydroxyurea, lovastatin, thymidine), early S (aphidicolin, cyclosporin A, rapamycin) or G2+M (colchicine, nocodazole) in 24 h cultures. Cytofluorimetric analyses showed that cycling Jurkat cells were equally distributed between the G1 (44.9 ± 6.5%) and S (42.3 ± 8.0%) phases. Cell distribution in G2+M was 12.7 ± 2.8%. Hydroxyurea but not lovastatin increased the percentage of cells in S phase to ≈60–70% and both drugs decreased it to ≈30% in G1. Thymidine had no effects. Aphidicolin increased the distribution in S phase to ≈70% with a decrease in G1 to ≈30%. Cyclosporin A and rapamycin increased the percentage of the cells in G1 to ≈70% and decreased it to ≈25% in S phase. Nocodazole increased cell distribution in G2+M to ≈60% and induced a decrease in G1 to ≈10%. The effects of the drugs were not related to their toxicity and their limited efficiency raised the possibility that Jurkat cells possessed an intrinsic resistance to these xenobiotics. Time-course analysis showed (scanning electron microscopy) that the early morphological changes induced by colchicine were reversible. Drug efflux experiments (vinblastine) suggested that an ATP-dependent process could be involved. However, Northern blot analyses showed a weak signal for MDR1 (P-glycoprotein). In contrast, a probe for MRP (P-190) showed a strong signal in Jurkat and peripheral lymphocytes. The presence of drugs (cyclosporin A, nocodazole, thymidine) (24 h) did not upregulate its message and cell treatment with -butathione (S,R)-sulfoximine only moderately affected the efficiency of the glutathione S-conjugate MRP transporter. Our data suggest that the intrinsic multidrug resistance of leukemic Jurkat T cells does not appear to involve the MDR1 and MRP members of the ABC family of reverse drug transporters and these observations raise the possibility of the involvement of multifaceted mechanisms.