姜黄素调节HL—60细胞周期蛋白表达的研究

目的探讨姜黄素对HL-60细胞周期的影响.方法选取HL-60细胞系,作细胞培养,采用SABC法测定分化抗原CDna和CD14阳性率,用TUNEL法判定细胞凋亡,用FCM法分析细胞周期,检测p27kipl、p21wafl、cyclinD3及pRbp-表达率.结果姜黄素呈时间及剂量依赖性抑制HL-60细胞增殖,用5、10、25μmol/L作用18h,增殖抑制率为4.7±1.2%、18.0±2.0%和37.5±2.4%;24h后抑制率达6.3±1.9%、25.0土2.2%和43.8±2.0%,可增加p27kip1、p21wafl表达,降低cyclinD3表达,使去磷酸化pRb(pRbp-)表达量增高;用10μmol/L姜黄素作用4d,p27kip1、p21waf1、pRbp-阳性率为47.8±3.6%、42.7±2.2%和23.5±1.5%,FCM进一步发现姜黄素作用后,p27kip1、cyclinD3荧光强度分别较对照组增高7倍及降低74.93%.细胞周期被阻滞于G0/G1和G2/M期,进而走向凋亡,且此能力呈时间及剂量依赖性.结论姜黄素能干扰细胞周期进程,诱导HL-60细胞凋亡,其作用机制与上调p27kip1、p21wa1及下调CyclinD3蛋白表达,增加pRb.表达水平,调控G1/S和G2/M期检测点功能有关.     

p27kip1基因诱导食管癌细胞凋亡实验研究

目的　探讨p2 7基因诱导人食管癌细胞凋亡的作用。方法　构建重组体腺病毒Ad p2 7kip1,转移到培养的人食管癌细胞EC 970 6 ,用流式细胞术、DNA片段分析法、TUNEL法观察Ad p2 7kip1诱导EC 970 6细胞凋亡的作用。 结果　成功构建重组体腺病毒Ad p2 7kip1,病毒滴度为 1.2 4× 10 12 CFU/ml,在感染倍数≥ 5 0感染强度时 ,即可达到 10 0 %的转导效率。Ad p2 7kip1转染食管癌细胞后 ,流式细胞术检测在G1期前出现亚倍体凋亡峰。细胞DNA抽提电泳后发现凋亡特征性梯度。TUNEL法检测凋亡指数分别为 37.3± 3.4 (Ad p2 7kip1组 )及 1.3± 0 .2 (空白对照组 ) ,差异有显著性(P <0 .0 1)。结论　p2 7可有效诱导食管癌细胞凋亡 ,这一发现将在探索食管癌的基因治疗方面具有重要意义     

姜黄素对血管平滑肌细胞增殖及cyclinD1和p21waf1/cip1蛋白表达的影响

目的 观察姜黄素（curcumin）抑制大鼠血管平滑肌细胞(VSMC)增殖和诱导细胞周期停滞的作用,以及对细胞周期蛋白cyclinD1,p21waf1/cip1表达的影响。方法 采用组织贴块法体外培养大鼠VSMC,MTT法测定姜黄素对VSMC增殖的抑制作用,流式细胞仪分析细胞周期分布,Western印迹法检测cyclinD1,p21waf1/cip1蛋白的变化。结果 MTT示不同浓度姜黄素(7.5～120 μmol/L)在24～72 h范围内,浓度和时间依赖性抑制VSMC增殖;30 μmol/L以上浓度姜黄素显著抑制增殖的VSMC细胞周期进程,使S及G2/M期减少(P<0.05),G0/G1期增加(P<0.05);30 μmol/L的姜黄素可抑制cyclinD1表达,促进p21waf1/cip1蛋白表达。结论 姜黄素具有明确的抑制VSMC增殖和细胞周期停滞的作用,其与cyclinD1,p21waf1/cip1蛋白变化有关。     

康莱特对Patu-8988细胞周期及其调节基因表达的影响

目的　研究康莱特注射液对人胰腺癌细胞周期及其调节基因表达的影响 ,探讨康莱特的药理机制。方法　通过流式细胞仪 DNA含量分析法检测康莱特对人胰腺癌细胞系 Patu- 8988细胞周期的影响 ,应用基因芯片技术分析加药前后细胞周期调节基因的表达差异 ,并以 Western blot对部分基因蛋白产物的表达进行验证。结果　 2 0 μl/ ml康莱特作用 Patu- 8988细胞 2 4 h后 ,G2 / M期细胞比率从(17.79± 0 .16 ) %上升至 (2 3.96 ± 2 .33) % ,而 S期细胞比率则由 (36 .6 1± 2 .97) %降至 (2 4 .76 ±4 .92 ) %。基因芯片结果显示 ,在 96条有关细胞周期的目的基因中共有 2 7条基因表达发生大于 3倍的显著变化 ,其中表达上调的基因 17条 ,下调的 10条。Western blot表明 cyclin A、cyclin B1、cyclin E、p2 1等蛋白表达改变与基因芯片结果一致。结论　康莱特的药理作用之一是使细胞周期相关基因的表达发生改变 ,从而影响细胞增殖。     

60Coγ射线分割照射对宫颈癌细胞周期及相关蛋白的影响

目的 观察不同剂量60Coγ射线分割照射对宫颈癌Hela细胞周期和细胞周期素(cyclin) D1、B1及细胞周期蛋白依赖激酶(CDK)1、4的影响.方法 0、2、4、6 Gy 60Co γ射线分割照射宫颈癌Hela细胞,1次/d,共5 d.用流式细胞术检测细胞周期和凋亡指数,RT-PCR测定cyclin D1、B1及CDK1、4.结果 2、4、6 Gy 60Co γ射线分割照射后各照射组G0/G1和G2/M期细胞数量逐渐增多,细胞凋亡指数随照射剂量增加而增加,与对照组相比,P均<0.05;cyclin D1、CDK1在所有照射组中均不表达,而对照组细胞正常表达;各照射组细胞cyclin B1和CDK4的表达与对照组相比,P均<0.05. 结论 2、4、6 Gy 60Co γ射线分割照射后Hela细胞阻滞在G0/G1、G2/M期,并诱导细胞凋亡,照射后细胞周期相关蛋白表达均受到抑制.     

胰腺癌p27kip1、视网膜母细胞瘤基因蛋白和增殖细胞核抗原表达及与临床病理关系的研究

目的　探讨细胞周期负性调控因子p2 7kip1,视网膜母细胞瘤 (Rb)基因蛋白和增殖细胞核抗原 (PC NA)在胰腺癌发生、发展中的作用。方法　应用免疫组织化学技术 (SP法 ) ,对 32例胰腺癌及癌旁组织中p2 7kip1、Rb蛋白和PCNA表达进行检测。结果　p2 7kip1蛋白阳性表达率在胰腺癌组织中为 5 6 2 5 %,显著低于癌旁胰腺组织 (84 37%) (P <0 0 5 ) ,并与胰腺癌组织分化程度及淋巴结转移相关 (P <0 0 5 ) ;Rb基因蛋白阳性表达率在胰腺癌组织中为 5 0 0 0 %,显著低于癌旁胰腺组织 (78 13%) (P <0 0 5 ) ;PCNA阳性表达率在胰腺癌组织中为 71 87%,显著高于癌旁胰腺组织 (4 3 75 %) (P <0 0 5 ) ,并与胰腺癌组织分化程度和淋巴结转移均相关(P <0 .0 5 )。结论　p2 7kip1、Rb基因蛋白和PCNA与胰腺癌发生、发展密切相关。     

支气管哮喘患者T淋巴细胞的细胞周期分布及周期调节蛋白表达的意义

目的通过检测T淋巴细胞的细胞周期分布及其细胞周期调节蛋白(CCRP)的表达,探讨发作期支气管哮喘(简称哮喘)患者T淋巴细胞过度活化、增殖的调控机制.方法采用碘化丙啶DNA染色法应用流式细胞仪分析30例发作期哮喘患者(哮喘组)及20名正常人(正常组)外周血T淋巴细胞的细胞周期分布;采用间接免疫荧光法,应用流式细胞仪同步测定相应T淋巴细胞内CCRP、依赖激酶抑制蛋白(P27kipl)、细胞周期蛋白E(cyclin E)、cyclin A、cyclin B的表达水平.比较哮喘患者和正常人上述指标的差异.结果哮喘组S期、S+G2/M期的T淋巴细胞百分率分别为(18±9)%和(25±10)%,对照组分别为(5±4)%、(11±6)%, 两组比较差异有显著性(P均<0.01);哮喘组G0/G1期的T淋巴细胞百分率为(76±10)%,对照组为(90±6)%,两组比较差异有显著性(P<0.01).哮喘组T淋巴细胞内P27kipl的表达水平为 (4.0±2.4)%,对照组为(6.7±4.8)%,两组比较差异有显著性(P<0.05);哮喘组cyclin E、cyclin A、cyclin B的表达水平分别为(25±24)%、(9±7)%、(6.4±5.9)%,对照组分别为(6±5)%、(4±4)%、(3.4±1.6)%,两组比较差异均有显著性(P均<0.01).结论 T淋巴细胞内CCRP异常表达与发作期哮喘患者T淋巴细胞过度活化、增殖有关,将CCRP作为调控靶点,可望成为治疗哮喘的新途径.     

食管癌细胞环氧合酶2表达与丝裂霉素C诱导有关

目的　探讨食管癌细胞环氧合酶 2 (COX 2 )表达与丝裂霉素C(MMC)处理的关系及可能机制。方法　四唑蓝法检测 2mg/LMMC处理对食管癌细胞株EC/CUHK 1生长的影响 ,流式细胞仪分析细胞周期及凋亡细胞比例 ,逆转录 PCR及Westernblot分别检测COX 2mRNA与COX 2、Bcl 2、p5 3及Rb蛋白的非磷酸化亚型 (pRb)表达。 结果　MMC处理后 0、0 5、2、4和 8h ,凋亡细胞比率分别为 (4 12± 0 83) % ,(1 0 0± 0 11) % ,(4 32± 0 99) % ,(9 4 6± 2 11) %和 (31 10±3 5 7) %。处理后 0 5、2和 4h ,COX 2mRNA相对表达量分别为处理前的 2 6 0、1 70和 0 0 8倍 ;COX 2蛋白为 2 0、3 1和 2 8倍 ;Bcl 2为 3 6、14 0和 12 0倍 ;p5 3为 1 8、0 5和 0 2倍 ;pRb为8 2、8 4和 6 2倍 ,Rb蛋白的磷酸化亚型 (ppRb)为 1 8、0 5和 0 2倍。MMC处理后COX 2mRNA、pRb和 p5 3蛋白之间以及COX 2、ppRb和Bcl 2蛋白之间的表达改变密切相关。 结论　MMC可上调食管癌细胞的COX 2表达 ,且明显与Bcl 2过表达相关 ,p5 3及Rb磷酸化积累可能调节COX 2蛋白的表达     

重组腺病毒介导的人p27kip1基因高表达对体外培养的大鼠主动脉血管平滑肌细胞迁移的影响

目的　探讨重组腺病毒介导的p2 7kip1基因及其蛋白产物高表达对血管平滑肌细胞 (VSMCs)迁移的抑制作用。方法　将含人p2 7kip1cDNA的重组腺病毒 (Adhp2 7kip1)及含 β 半乳糖苷酶基因的重组腺病毒 (AdLacZ)在体外转染原代大鼠主动脉VSMCs,用Westernblot及Boyden趋化小室检测外源性p2 7kip1蛋白在细胞内的表达及对VSM Cs迁移的影响。结果　转染后 2 4h ,Adhp2 7kip1转染的VSMCs内p2 7kip1蛋白高表达 ,而AdLacZ转染的VSMCs内仅显示极低水平的内源性p2 7kip1蛋白表达 ;Boyden趋化小室检测显示未转染的VSMCs、AdLacZ及Adhp2 7kip1转染的VSMCs血清诱导后的迁移细胞数分别为 139± 2 6、10 6± 16及 6 8± 14。结论　外源性p2 7kip1基因及蛋白产物在VSMCs内高表达可显著抑制VSMCs的迁移     

阻断泛素-蛋白酶体通路对胃癌细胞增殖和凋亡的影响

目的　研究阻断泛素 蛋白酶体通路对胃癌细胞增殖的抑制作用及其机制。方法　将泛素 蛋白酶体通路特异性阻断剂MG 132加入胃癌细胞株SGC 790 1,四甲基偶氮唑蓝 (MTT)法测定细胞抑制效应 ,流式细胞仪 (FCM )检测细胞周期及凋亡 ,DNA片段分析进一步证实凋亡的存在 ,TRAPPCR ELISA法检测端粒酶活性 ,免疫细胞化学检测 p2 7kip1的表达。 结果　MG 132对SGC 790 1细胞有显著抑制作用 ;FCM显示对照组胃癌细胞G0 /G1期比例为 (46 .3± 4 .1) % ,MG 132作用后为 (72 .1± 5 .0 ) % ,较对照组增加 (P <0 .0 1) ,并有明显的亚二倍体凋亡峰 ;细胞DNA抽提电泳后发现特征性凋亡梯状条带 ,TRAPPCR ELISA法检测示MG 132作用胃癌细胞 2 4、4 8、72、96h时A值分别为 0 .197± 0 .0 0 7、0 .0 81± 0 .0 0 5、0 .0 74± 0 .0 0 4、0 .0 6 3± 0 .0 0 2 ,对照组A值分别为 1.80 1± 0 .0 4 8、1.887± 0 .0 72、2 .0 4 7± 0 .0 85、2 .131± 0 .0 76 ;端粒酶活性显著受抑制 (P <0 .0 1) ;p2 7kip1在胃癌细胞中为胞质表达 ,经MG 132作用后 ,胞质、胞核均有表达。结论　MG 132能显著抑制胃癌细胞株SGC 790 1的增殖、诱导其凋亡 ,其机制是增强 p2 7kip1表达 ,使细胞产生G1期阻滞 ,并抑制端粒酶活性。     

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.     

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.     

Germ cell stimulation of Sertoli cell protein phosphorylation

Cultures of Sertoli cells were treated with freshly isolated, intact germ cells to determine if germ cells were capable of influencing Sertoli cell function. By using two-dimensional polyacrylamide gel electrophoresis and autoradiography, germ cells were found to increase several-fold the incorporation of [32P]orthophosphate into two phosphoproteins (which we term germ cell-dependent phosphoproteins 1 and 2 or GC1 and GC2) of Sertoli cells. Increased phosphorylation of GC1 and GC2 was rapid, germ cell dose dependent, and calcium dependent. The increased phosphorylation of GC1 appears to involve calmodulin-dependent protein kinase, while phosphorylation of GC2 appears to involve the activation of calcium/phospholipid-dependent protein kinase (protein kinase C). Medium conditioned by germ cells was capable of eliciting the same response in Sertoli cells. Germ cells had no effect on the phosphorylation of proteins in Chinese hamster ovarian cells, but did result in increased phosphorylation of a protein in TR-ST cells, which migrated similarly to GC1 of Sertoli cells. Neither Chinese hamster ovarian nor TR-ST cells had any effect on Sertoli cell protein phosphorylation. These results indicate that germ cells may be directly involved in the local regulation of Sertoli cell function within the seminiferous epithelium. The results further suggest that the mechanism of germ cell-Sertoli cell interaction involves the mobilization of intracellular calcium, activation of Ca2+/calmodulin-dependent protein kinase, and protein kinase C. We infer from these results that germ cell-Sertoli cell interaction may operate via hydrolysis of Sertoli cell membrane phophatidylinositols.     

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.     

Red cell rheology in stomatocyte-echinocyte transformation: roles of cell geometry and cell shape

The influence of the shape of the red blood cell during stomatocyte- echinocyte transformation on its deformability was studied by microsieving through pores with diameters of 2.6, 4.5, and 6.9 micron. A stomatocytic transformation was produced by chlorpromazine (0.02, 0.1, and 0.5 mmol/L) and an echinocytic transformation by sodium salicylate (7.5, 30, and 120 mmol/L). For spherostomatocytes, an increase in filtration resistance through 2.6 and 4.5 micron pores was observed, whereas for spheroechinocytes, a decrease in filtration resistance through 2.6 micron pores was found. Larger pores (6.9 micron) were not sensitive to those shape changes. The changes in deformability can be explained by the fact that the surface area of (sphero)-stomatocytes decreased, whereas that of (sphero)-echinocytes increased; the cell volume remained essentially constant. Echinocytes produced by 24-hour adenosine triphosphate depletion differed from drug- induced echinocytes: they had an increased cell volume at constant surface area and consequently an increased filtration resistance through 2.6- and 4.5-micron filter pores. Shape changes with spicule formation are therefore not a homogeneous entity, and cell geometric factors (eg, surface area and volume) must be assessed with care. The viscosity of red cell suspensions at a hematocrit level of 45% was higher for drug-induced echinocytes than discocytes or stomatocytes at all shear rates tested. We conclude that the normal discocyte represents an optimum shape for the flow in vivo since a stomatocytic transformation could impair the passage through the microcirculation (decrease in cell filterability) and an echinocytic transformation could impair the flow in larger vessels (increase in blood viscosity).     

Red cell magnesium as a function of cell age

The variation in the magnesium content of human red cells as a function of cell age has been measured by atomic absorption spectrophotometry. The cell population was split into different age fractions using discontinuous density gradient centrifugation, since it is known that cell density increases with age. A mathematical model relating predicted cell age to cell density has been developed which allows the quantification of the observed fall-off in magnesium content with cell age. This model suggests that cells lose magnesium monoexponentially with age, the half-life being approximately 100 days. A previously proposed hypothesis that magnesium could enter the cells only at erythropoiesis and then decay monoexponentially predicted a half-life of 22.4 days and is therefore seen to be an oversimplification of magnesium kinetics in the red cell. The relevance of the present findings to pathologic conditions with abnormal red cell magnesium concentrations is discussed.     

Kinetics of dendritic cell chimerism and T cell chimerism in allogeneic hematopoietic stem cell recipients

Dendritic cells (DC) as potent antigen-presenting cells (APC) and T cells as effector cells play an essential role in the pathophysiology of both graft-versus-host (GvH) and graft-versus-leukemia (GvL) reactions after transplantation. Therefore, we determined the kinetics of DC and T-cell chimerism establishment after allogeneic hematopoietic cell transplantation (AHCT) in a group of 144 patients, using fluorescence-activated cell sorting (FACS) or magnetic cell sorting (MACS) followed by FISH or STR-PCR analysis for chimerism evaluation. In all, three cell lines investigated (CD3(+) T cells, CD11c(+) DC1 and CD123(+) DC2), we found a rapid and consistent establishment of complete donor chimerism (CDC) in over 70% of all patients during the first 6 weeks after AHCT. The rate of patients with CDC increased significantly over time within the first year after transplantation. A related donor (P=0.004) as well as an underlying lymphatic leukemia (P=0.03) were found to be significantly associated with development of MC in T cells. No significant correlation between DC or T cell chimerism and GvHD or relapse was detected. Our results thus demonstrate a fast and stable CDC in DC1, DC2 and T cells after AHCT that continuously increases over time in nearly all patients.     

Establishment and characterization of a mantle cell lymphoma cell line

A mantle cell lymphoma (MCL) cell line (JeKo-1) was established from peripheral blood mononuclear cells of a patient with a large cell variant of MCL showing leukaemic conversion. JeKo-1 cells were Epstein-Barr virus negative and showed a B-cell phenotype with IgM⁺, IgD⁺, CD3^?, CD5⁺, CD10^?, CD19⁺, CD20⁺ and CD23^?; they overexpressed cyclin Dl, Bcl-2, c-Myc and Rb proteins. Bcl-1/J_H gene rearrangement was confirmed by polymerase chain reaction, although karyotypic analysis showed 40/41 chromosomes devoid of apparent t(11;14)(q13;q32) translocation. JeKo-1 cells were highly tumourigenic in SCID mice.