存活素反义寡核苷酸对甲状腺癌细胞生长的抑制作用

目的 探讨利用凋亡抑制基因存活素反义寡核苷酸（ASODN）诱导甲状腺癌细胞凋亡的效应。方法 设计合成特异性靶向存活素的ASODN，以不同浓度和时间对人甲状腺髓样癌细胞进行转染，并设空白、正义（SODN）对照组进行比较。采用RT-PCR、Western印迹、免疫细胞化学技术检测各组细胞中存活素mRNA、蛋白表达水平，DNA裂解分析、吖啶橙／溴化乙锭（AO／EB）染色法、流式细胞仪检测细胞凋亡水平和形态变化，MTT法检测细胞生长抑制情况。结果 转染ASODN各组细胞存活素mRNA和蛋白表达均较空白对照组、SODN组明显减弱；细胞凋亡率显著增加，细胞生长相应受抑，上述效应在ASODN转染24h最为明显，并呈浓度依赖性；而各时段SODN组与空白对照组间各项指标差异均无统计学意义。结论 ASODN能够特异性地下调甲状腺癌细胞中存活素基因表达，进而诱导肿瘤细胞凋亡，并抑制其增殖。     

热休克蛋白70通过Bcl-2抑制氧化应激所致C2C12细胞凋亡

目的 探讨C2C12肌原细胞内热休克蛋白70对Bcl-2表达的影响及Bcl-2对热休克蛋白70抗细胞凋亡作用的影响.方法 应用Western Blotting观察Bcl-2在转染热休克蛋白70真核表达质粒(pcDNA3.1-HSP70)或其反义寡核苷酸C2C12肌原细胞中的表达;采用基因瞬间转染技术使热休克蛋白70过表达的C2C12肌原细胞内Bcl-2表达抑制,应用流式细胞术检测H2O2处理所致细胞凋亡的发生情况.结果 转染热休克蛋白70真核表达质粒的C2C12肌原细胞中,热休克蛋白70和Bcl-2的表达明显高于空载体转染组(P<0.01);而转染热休克蛋白70反义寡核苷酸后,热休克蛋白70和Bcl-2的表达明显低于随机寡核苷酸转染组 (P<0.01);热休克蛋白70过表达的C2C12肌原细胞分别转染Bcl-2反义寡核苷酸及随机寡核苷酸,0.5 mmol/L H2O2处理24 h,Bcl-2反义寡核苷酸转染组的细胞凋亡率明显高于随机寡核苷酸转染细胞组.结论 热休克蛋白70能上调C2C12肌原细胞内Bcl-2的表达;热休克蛋白70的抗细胞凋亡功能可能与其上调Bcl-2表达相关.     

信号转导子和激活子3通路调控大鼠血管平滑肌细胞增殖迁移机制研究

目的研究转录信号转导子和激活子3（stat3）通路与大鼠血管平滑肌细胞（VSMCs）增殖迁移的关系,明确VSMCs增殖的信号转导过程。方法应用脂质体转染Stat3反义寡核苷酸作用于大鼠VSMCs,应用酶联免疫法（ELISA）检测Stat3水平变化及MTF法检测细胞增殖状态,蛋白免疫印迹法（Western blot）检测stat3、磷酸化stat3及其靶基因产物Cyclin D1、Bcl—XL的表达。结果转染Stat3反义寡核苷酸后,大鼠VSMCs中Stat3水平明显下降（P〈0．01）,同时其增殖水平降低,而相应空白对照组、脂质体组、转染正义寡核苷酸组变化不明显。转染Stat3反义寡核苷酸的VSMCs中stat3、p-Stat 3、Cyclin D1蛋白表达水平随作用时间延长而下降（P〈0．01）,而Bcl—xL水平无明显变化。结论癌基因stat3信号通路与大鼠VSMCs增殖高度相关,可能通过其下游靶基因Cyclin D1影响其增殖,而阻断此通路则可抑制VSMCs的增殖。     

氯通道ClC-3反义寡核苷酸对过氧化氢诱导的大鼠主动脉平滑肌细胞凋亡的影响

目的研究ClC3反义寡核苷酸对H2O2诱导的大鼠主动脉平滑肌细胞凋亡的影响。方法蛋白免疫印迹法检测ClC3蛋白表达;形态学方法、DNA琼脂糖电泳、MTT法和流式细胞仪观察和分析H2O2诱导的大鼠主动脉平滑肌细胞形态学改变、DNA断裂、细胞存活率和凋亡率及ClC3反义寡核苷酸转染对其影响。结果ClC3反义寡核苷酸转染抑制内源性ClC3蛋白表达后,可加重H2O2诱导大鼠主动脉平滑肌细胞形态学改变及DNA断裂,细胞凋亡率由52.8%±13.6%增至75.7%±5.8%(n=6,P<0.01),而细胞存活率由48.9%±4.3%进一步降低为31.3%±4.3%(n=6,P<0.01)。结论ClC3反义寡核苷酸转染促进H2O2诱导的大鼠主动脉平滑肌细胞凋亡。     

转化生长因子β1反义寡核苷酸抑制单侧肾切除糖尿病大鼠系膜增生

目的 探讨转化生长因子（TGF）β1反义寡核苷酸对单侧肾切除糖尿病大鼠系膜增生的作用。方法 采用人工合成TGF－β1反义、正义及错配的3组寡核苷酸经脂质体包裹柏转染单侧肾切除糖尿病大鼠肾小球系膜细胞（MC）。用细胞计数检测转染后MC的生长抑制率，用RT－PCR和ELISA方法检测TGF－β1 mRNA和蛋白的表达水平，并检测IV型胶原mRNA表达水平的变化。结果 TGF－β1反义寡核苷酸可抑制MC的存活，TGF－β1 mRNA及蛋白的表达水平下降，IV型胶原mRNA的水平也下降。结论 TGF－β1反义寡核苷酸不仅抑制TGF－β1 mRNA及蛋白的表达，而且还可抑制细胞外基质IV型胶原mRNA的表达，抑制MC的肥大，从而抑制单侧肾切除糖尿病大鼠系膜的增生。     

反义c-myc寡核苷酸对骨肉瘤细胞MG-63增殖的影响及机制

目的探讨反义c-myc寡核苷酸对人骨肉瘤MG-63细胞增殖的影响及机制。方法采用反义核酸技术设计反义c-myc寡核苷酸片段,转染入人骨肉瘤MG-63细胞,MTT法及流式细胞仪法检测瘤细胞体外增殖、细胞周期及c-myc基因蛋白表达情况。结果MTT法示人骨肉瘤MG-63细胞的增殖受抑,10．0μmol／L、作用48h效果最明显;流式细胞仪示反义c—myc寡核苷酸主要阻止人骨肉瘤MG-63细胞进入S期,c-myc基因蛋白表达受抑。结论反义c-myc寡核苷酸可抑制人骨肉瘤MG-63细胞的增殖;其机制为在基因水平干扰c-myc基因蛋白的表达。推测用反义策略抑制癌基因表达,或用基因敲除、基因替换的方法去除癌基因,可达到基因治疗的目的。     

反义EGFR寡核苷酸对AngⅡ促血管平滑肌细胞增殖效应的影响

目的探讨脂质体介导的反义表皮生长因子受体（EGFR）寡核苷酸基因转染对血管紧张素Ⅱ（AngⅡ）促大鼠血管平滑肌细胞（VSMC）增殖的影响。方法用EGFR寡核苷酸脂质体复合物转染Sprague-Dawley大鼠VSMC,通过RT-PCR、Western-Bloting分别检测转染后EGFR mRNA及蛋白的表达情况,用3H-Tdr掺入法检测经EGFR寡核苷酸转染再用AngⅡ刺激VSMC的增殖情况。结果反义EGFR寡核苷酸转染大鼠VSMC后,EGFR mRNA及蛋白的表达较正义组及对照组显著减少（P<0.01）;用AngⅡ刺激后,反义组细胞的3H-Tdr掺入较正义组及对照组显著降低（P<0.01）。结论脂质体介导的反义EGFR寡核苷酸转染可以减弱AngⅡ的促VSMC增殖效应。     

VEGF反义寡核苷酸对体外生长的大肠癌HT-29细胞的抑制作用

目的:探讨血管内皮生长因子(VEGF)反义寡核苷酸(ASODN)对体外生长的人大肠癌HT-29细胞的抑制作用.方法:实验设空白对照组、脂质体转染组、错义链转染组(SODN组)和不同浓度反义链转染组(ASODN组).用LipofectamineTM2000介导的VEGF ASODN和错义寡核苷酸(SODN)转染人大肠癌细胞株HT-29,半定量RT-PCR检测各组细胞VEGF mRNA的表达:Western blot测定转染48、72 h后VEGF蛋白表达:MTT法和流式细胞术检测细胞增殖和凋亡.结果:转染48 h后,ASOND组的VEGF mRNA表达水平明显低于脂质体对照组和SODN组(0.455±0.032 vs 0.934±0.031,0.915±0.004,p<0.01);脂质体对照组与SODN组之间无显著差异.细胞转染48、72 h后,ASODN组蛋白表达明显弱于脂质体对照组和SODN组,且72h弱于48 h.与对照组比较.VEGF ASODN对HT-29细胞有明显的生长抑制作用,并且抑制呈剂量和时间依赖性(P<0.05).结论:VEGF ASODN通过抑制VEGF的基因表达,对体外生长的人大肠癌HT-29细胞的增殖进行抑制.     

人宫颈癌基因反义核酸抑制肝癌细胞生长作用的实验研究

目的探讨针对人宫颈癌基因(HCCR)反义核苷酸影响肝癌细胞增殖、凋亡的作用。方法构建pcD-NA3.1-HCCR反义真核表达质粒,转染HepG2细胞,实时定量PCR检测HCCR mRNA表达水平,Western blot检测其蛋白表达水平,流式细胞仪观察HepG2细胞凋亡和细胞周期的变化,MTT法检测细胞的增殖活性。结果HCCR反义核酸可有效抑制HepG2细胞HCCR的表达,与转染前相比,转染后24小时HCCR mRNA水平下降到16%,其蛋白表达水平在24小时也明显降低。对细胞的增殖与凋亡活性检测显示:转染HCCR反义质粒后,HepG2细胞增殖受到抑制,转染后24小时抑制率为47.62%(P<0.05),细胞凋亡率为14.34%±0.91%,较对照组明显增多(t=21.799,P<0.05),细胞分裂多停止在G0G1期。结论HCCR反义核酸可以抑制HepG2细胞中HCCR的表达,抑制细胞增殖,促进细胞凋亡,使细胞停滞于G1期。HCCR反义核酸用于肝癌细胞的基因治疗具有一定的潜在意义。     

Bcl-2反义核酸的设计及对K562细胞凋亡的研究

目的：探讨bcl-2不同靶点的反义寡核苷酸（ASODN）对白血病细胞株K562细胞凋亡的影响。方法：用RNA二级结构预测程序预测bcl-2mRNA的二级结构；免疫荧光标记观察细胞bcl-2蛋白水平；细胞记数观察细胞的生存情况；用流式细胞仪观察细胞凋亡。结果：在5个初选的反义序列中，靶向bcl-2mRNA蛋白编码区和翻译起始区的ASODN能明显地下调bcl-2蛋白的表达，并且两个不同靶点的ASODN能有效地抑制K562细胞的生长活性、促进细胞凋亡；靶向bcl-2mRNA蛋白编码区的ASODN降低细胞bcl-2蛋白、促进细胞凋亡的作用较靶向翻译起始区的ASODN强。随机的无诳寡核苷酸对K562细胞的生长活性、bcl-2蛋白水平及细胞凋亡率均无影响。结论：分别在bcl-2mRNA翻译起始区和蛋白编码区发现了两个有效的反义作用靶点，针对这两个靶点的ASODN能特异性促进K562细胞的凋亡。     

Liver cell adenoma and liver cell adenomatosis

During the last three decades liver cell adenoma and liver cell adenomatosis have emerged as new clinical entities in hepato-logical practice due to the widespread use of oral contraceptives and increased imaging of the liver. On review of published series there is evidence that 10% of liver cell adenomas progress to hepatocellular carcinoma, diagnosis is best made by open or laparoscopic excision biopsy, and the preferred treatment modality is resection of the liver cell adenoma to prevent bleeding and malignant transformation. In liver cell adenomatosis, the association with oral contraceptive use is not as high as in solitary liver cell adenomas. The risk of malignant transformation is not increased compared with solitary liver cell adenomas. Treatment consists of close monitoring and imaging, resection of superficially located, large (>4 cm) or growing liver cell adenomas. Liver transplantation is the last resort in case of substantive concern about malignant transformation or for large, painful adenomas in liver cell adenomatosis after treatment attempts by liver resection.     

Red cell exchange in sickle cell disease

Red cell exchange transfusion is frequently of use in the management of patients with sickle cell disease either electively or therapeutically. Modern cell separators allow this procedure to be performed rapidly, effectively and safely. These machines have a number of advantages over manual exchange procedures. The patient remains isovolaemic, there is little loss of plasma or platelets, the procedure is relatively short and in elective circumstances can be performed on an outpatient basis. In this series 66 exchanges were performed on 21 patients with an overall increase in HbA of 70%. The COBE Spectra gave a mean increase in HbA of 77%, with the majority of patients achieving an HbA of > 90% post exchange. Automated redcell exchange was well tolerated by most patients, and adverse effects were limited to symptoms of hypocalcaemia which were easily treated, and to transfusion reactions. Cell separators can therefore be recommended for exchange transfusion in patients with sickle cell disease, who require an increase in HbA levels either prophylactically or therapeutically. They are safe, effective, easy and quick to use.     

Erythrocyte-endothelial cell adherence in sickle cell disorders

Detachment of individual sickle erythrocytes from cultured endothelial cell monolayers has been evaluated by a fluid-shearing technique in an effort to quantitate adherence at shear forces that would be anticipated in the in vivo circulation. Nonirreversibly sickled cells (non-ISC) were more adherent at normal oxygen tensions than control cells. More than 1% non-ISC remained attached to the monolayer at forces greater than physiologic shear stresses in capillary and venous circulations, and many of the most avidly attached cells, once separated, immediately reattached to adjacent endothelial cells. These data suggest that hemoglobin S-containing erythrocytes may have a higher frequency of adherence in vivo in regions of low shear stress where prolonged erythrocyte-endothelial cell contact could occur. Some of these cells detached by shear force would subsequently reattach in in vivo conditions. Plasma-enhanced attachment frequency and plasma from blood in a case of sickle crisis caused further increase. These observations further support the concept that sickle erythrocyte- endothelial cell interaction may be a significant factor in initiation of vascular occlusive events in sickle cell disease.     

Actin filament debranching regulates cell polarity during cell migration and asymmetric cell division

The formation of the branched actin networks is essential for cell polarity, but it remains unclear how the debranching activity of actin filaments contributes to this process. Here, we showed that an evolutionarily conserved coronin family protein, the Caenorhabditis elegans POD-1, debranched the Arp2/3-nucleated actin filaments in vitro. By fluorescence live imaging analysis of the endogenous POD-1 protein, we found that POD-1 colocalized with Arp2/3 at the leading edge of the migrating C. elegans neuroblasts. Conditional mutations of POD-1 in neuroblasts caused aberrant actin assembly, disrupted cell polarity, and impaired cell migration. In C. elegans one-cell−stage embryos, POD-1 and Arp2/3, moved together during cell polarity establishment, and inhibition of POD-1 blocked Arp2/3 motility and affected the polarized cortical flow, leading to symmetric segregation of cell fate determinants. Together, these results indicate that F-actin debranching organizes actin network and cell polarity in migrating neuroblasts and asymmetrically dividing embryos.

Cell polarity is a fundamental feature of virtually all eukaryotic cells and plays crucial roles in a wide range of cellular processes, including cell motility, asymmetric cell division, and cell signaling (1). The establishment of cell polarity involves the asymmetric assembly of distinct cellular components to perform specialized functions. The actin-related protein (Arp) 2/3 complex-dependent branched actin networks and the pushing force they produce provide the principal means for cells to remodel the plasma membrane during cellular polarization (2). For example, in the leading edge of a migrating cell, the local Arp2/3-nucleated actin polymerization powers asymmetric projections of the plasma membrane (3). During asymmetric cell division of the Caenorhabditis elegans zygote, an actomyosin flow is central to the transport of the polarity PAR proteins into defined subcellular domains (4).Actin filaments'' continuous assembly must be balanced by actin depolymerization to ensure a constant supply of actin monomers for new growth. The Arp2/3 complex potency in actin nucleation empowers this complex as an essential regulator to organize the actin cytoskeleton. While Arp2/3 by itself is biochemically inactive, interactions with nucleation-promoting factors (NPFs) such as the Wiskott Aldrich syndrome protein (WASP)/WASP family verproline-homologous (WASP/WAVE) family proteins shift the Arp2/3 complex from its open, inactive conformation to a closed, active conformation (5, 6). The conformationally activated Arp2/3 complex then binds to the side of preexisting actin filaments to nucleate a branch from the mother filament (7–12). Conversely, nucleation by Arp2/3 can be inhibited by several binding partners, including glia maturation factor (GMF), Gadkin, Arpin, and Coronin, whose activities replenish available pools of actin monomers and Arp2/3 complexes for sustained actin assembly (13–18).The coronin family proteins are conserved actin regulators (19). The phylogenetic analysis grouped coronin genes into three types (19, 20). The best-characterized coronin is the Type I coronin (e.g., Coronin 1B) that binds to actin filaments through the β-propeller structure and to the Arp2/3 complex via its N terminus. These interactions block the docking of Arp2/3 onto actin filaments or facilitate debranching the existing actin network (20). Coronin 1B simultaneously interacts with the Slingshot phosphatase to dephosphorylate and activate ADF/Cofilin proteins that sever actin filaments, thereby promoting the actin network disassembly (13). Despite significant progress on Type I coronin, the activity and function of other coronins remain unclear. In particular, Type III coronins, known as POD-1 in C. elegans and Drosophila or Coronin7 in Dictyostelium and humans, contain two tandem coronin repeats, making them distinct from other coronins (19–21). POD-1 was biochemically isolated from C. elegans oocytes (22), and its mutations disrupted the polarity and architecture in early C. elegans embryos and impaired midlife touch sensitivity of the nematode (21, 23). However, it remains unclear how the Type III coronin functions. The Drosophila homolog of POD-1 is required for correct axon guidance, and the purified Dpod-1 cross-links the actin and microtubule cytoskeletons (24), whereas the mammalian Coronin7 was implicated in the Golgi morphology and function (25, 26), demonstrating the functional divergence of this family of coronin. Here, we show that the C. elegans POD-1 debranches Arp2/3-nucleated actin filaments in vitro and that POD-1 regulates cell polarity by remodeling the actin cytoskeleton during cell migration and asymmetric cell division.     

Red cell alloimmunization in sickle cell disease

Alloimmunization to red cell antigens contributes to morbidity in transfused patients. It has been recommended that blood for sickle cell patients need not be matched for antigens other than ABO and Rh(D), as there is no greater incidence of antibody production than in other multitransfused patient populations. Post transfusion alloimmunization was studied in a group of 34 sickle cell disease patients attending a U.K. haemoglobinopathy clinic. Red cell antibodies were formed in 17.6% of the transfused patients and Rhesus and Kell antibodies accounted for 66% of this total. In order to reduce alloimmunization, a policy of performing extended red cell phenotyping on the patients, and providing blood matched for Kell, and in certain circumstances the Rhesus antigens other than Rh(D), is recommended.     

Misdiagnosis of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (RCC) represents the most common histological subtype of malignant kidney tumors. Based on symptoms alone, clear cell RCC is indistinguishable from other histological classes of RCC unless the tumor is present in the context of an RCC syndrome. Histopathological examination is, therefore, important to accurately identify clear cell RCC. Clear cell RCCs have characteristic morphological criteria; these tumors can be easily identified upon typical presentation, but diagnosis can be challenging when tumor cell pattern is unusual or when availability of tissue samples is limited. In this Review, the clinical, radiological and pathological characteristics of clear cell RCCs are described, as well as the potential tumors that can be confused with clear cell RCC and need to be considered in the differential diagnoses. Finally, the importance of an accurate diagnosis is highlighted in the context of the increasing use of preoperative tissue sampling and the prevalence of clear cell tumors associated with hereditary syndromes, which could have different therapeutic and prognostic implications for patients and their families.     

Mechanism of red blood cell aging: Relationship of cell density and cell age

The human red cell has a life span of 120 days. The mechanism that determines cell removal from the circulation with such precision remains unknown. Most studies of red cell aging have been based on analysis of cells of progressively increasing age separated by density. The relationship between red cell age and density has been recently challenged, and the hypothesis has been put forward that cell death is not the result of a progressive deterioration of essential cell constituents. This theory was based on preliminary observations in transient erythroblastopenia of childhood, which could not later be confirmed. When the relationship between cell aging and increasing density is critically reviewed, it appears to be based on firm experimental evidence, confirmed by in vivo demonstration of decreasing survival of cells of increasing age. Analysis of studies using buoyant density gradients reveals that this technique can easily distinguish the single exponential slope of decline for those cell components that change progressively throughout the red cell life span from the biphasic decline of those that decrease drastically at the reticulocyte-mature red cell transition. The view that the aging of the red cell and its removal from the circulation result from a progressive series of events during the 120 days of its life span appears to be the most consistent with the available data. Density separation, validated by much experimental evidence, remains a most useful technique for the study of the mechanism of aging of the red cell. © 1993 Wiley-Liss, Inc.     

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

Background

Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs.

Methods

Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs.

Results

The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability.

Conclusions

Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.     

Automated red cell exchange in sickle cell disease

We used multiple optical trapping to study the mechanism of red cell (dis)aggregation. Two sets of optical 'tweezers' were used to bring two red blood cells together to form a two-cell aggregate and then to pull them apart, to study the interaction between the cells.
We found that cross-bridging occurred in normal reversible aggregation as we observed binding and the occurrence of small tethers between opposite cell membranes. Furthermore, the cells could only be parted by sliding them side by side with a maximum velocity in the order of μm/s indicating accumulation of the cross-bridges.     

胆红素对肝星状细胞-T6增殖及细胞周期的影响

[目的]观察胆红素对肝星状细胞(HSC)-T6增殖及细胞周期影响.[方法]将培养细胞分成正常组和胆红素不同浓度(10 μmol/L、30 umol/L、50 /μmol/L、70 μmol/L、100 μmol/L)干预组,采用MTT法观察胆红素对HSC-T6增殖的影响,流式细胞仪观察各组细胞周期的变化.[结果]①不同浓度胆红素对HSC-T6均有促进增殖作用,且呈一定的量效关系,与正常组比较差异有统计学意义(P＜0.05);②10 μmol/L、50 μmol/L、100 μmol/L浓度胆红素作用HSC-T6后,G0/G1期减少,S期增加,G2/M期增加,与正常组比较均P＜0.05.[结论]胆红素对HSC-T6均有促进增殖作用.