白藜芦醇诱导白血病Molt-4细胞凋亡及对WAVE1基因表达的影响

目的 探讨白藜芦醇诱导人类急性淋巴细胞白血病Molt-4细胞凋亡的作用机制.方法 噻唑蓝比色法(MTF)测定细胞生长抑制率;流式细胞术检测细胞周期分布及凋亡率;半定量反转录聚合酶链反应(RT-PCR)法检测WAVE1基因的表达.结果 MTT结果显示:12.5、25.0、50.0、100.0、200.0 μmol/L的白藜芦醇作用于Moh-4细胞24、48、72 h后,细胞的最大抑制率分别达到29.32%、36.11%、53.92%、62.50%、74.98%,并呈时间-剂量依赖性(F=33.614,P<0.05);流式细胞术检测结果显示:与对照组相比,50.0、100.0 μmol/L白藜芦醇作用于Molt-4细胞48 h后,S期细胞比例由42.2%分别上升为68.6%和78.1%,细胞发生了S期阻滞(F=19.453,P<0.01);PCR结果显示:50.0、100.0 μmol/L的白藜芦醇处理Molt-4细胞48 h后,WAVE1/GAPDH比值分别为0.356±0.03、0.382±0.05,与对照组(0.586±0.06)比较,差异有统计学意义(F=8.950,P<0.01).结论 白藜芦醇可诱导Moh-4细胞发生凋亡,其作用机制可能与下调WAVE1基因表达有关.     

姜黄素联合人类细胞因子诱导的杀伤细胞对卵巢癌细胞增殖的抑制作用及其机制

目的:观察姜黄素(Cur)联合人类细胞因子诱导的杀伤(CIK)细胞在体外对人卵巢癌浆液性囊腺癌细胞SKOV-3的增殖抑制作用,探讨其协同抗肿瘤作用及其可能机制.方法:诱导脐血CIK细胞,将SKOV-3细胞随机分为Cur组、CIK细胞组和Cur联合CIK细胞组,MTT法测定各组细胞的增殖抑制率,RT-PCR检测各组细胞Fas相关死亡结构蛋白Fas、Fas相关死亡结构蛋白(FADD)和Caspase-3 mRNA的表达.结果:与Cur组和CIK细胞组比较,Cur联合CIK细胞组SKOV-3细胞增殖抑制率增大,并且随时间的延长或剂量的增加,增殖抑制率亦增加,在效靶比为12.5∶1,20 μmol·L-1 Cur作用48 h时,SKOV-3细胞增殖抑制率达到76.2%;Cur联合CIK细胞组与Cur组和CIK细胞组比较,SKOV-3细胞Fas及Caspase-3的mRNA表达水平增加(P<0.05),Cur组、CIK细胞组和Cur联合CIK细胞组SKOV-3细胞FADD-mRNA表达无明显变化.结论:CIK细胞与Cur合用具有协同抗肿瘤作用,其效应可能与促进SKOV-3细胞Fas及Caspase-3的mRNA表达有关.     

五味子多糖对甲状腺癌细胞株SW579凋亡及survivin表达的影响

目的:探讨五味子多糖对人甲状腺癌SW579细胞株的生长抑制作用及其机制.方法:48只成年雄性Wistar大鼠分为阴性对照组和药物组,阴性对照组予以生理盐水,药物治疗组予以不同剂量的五味子多糖,分别提取其血清.SW579细胞分为空白组、阴性对照组和药物组.空白组为15%小牛血清的RPMI-1640培养液,阴性对照组细胞加入阴性组大鼠的纯化血清,药物组分别加入不同浓度(100、200 和 400 mg·kg-1)的含药血清,AO/EB荧光染色和流式细胞仪检测细胞凋亡情况,MTT法检测细胞增殖情况,Western blotting检测细胞survivin的表达.结果:流式细胞术结果显示,低、中和高剂量药物组细胞凋亡率为8.63%、35.63%和38.32%,明显高于空白对照组和阴性对照组细胞凋亡率(4.23%和4.10%)(P<0.01),并随剂量增加而增加.阴性对照组细胞生长抑制率为(0.12±0.06)%,低、中和高剂量药物组细胞生长抑制率为(27.51±1.62)%、(34.69±0.79)% 和(45.83±1.33)%,药物组与阴性对照组比较差异有统计学意义(P<0.01).Western blotting 检测低、中、高剂量药物组细胞survivin的表达明显低于空白对照组和阴性对照组(P<0.01).结论:五味子多糖能有效抑制人甲状腺癌细胞株SW579中survivin基因表达,诱导SW579细胞凋亡,抑制肿瘤细胞增殖.     

富血小板血浆对体外培养条件下人真皮成纤维细胞增殖能力的影响

目的:探讨富血小板血浆(PRP)对人真皮成纤维细胞(hDFbs)在体外培养条件下增殖能力的影响,探讨PRP促进皮肤、黏膜伤口愈合的机制.方法:PRP和hDFbs来源于健康成年人,两次离心法制备PRP,倒置相差显微镜观察0、12.5%、25.0%、50.0%和100.0%PRP浓度作用下 hDFbs的增殖;免疫细胞化学检测50.0%浓度不同剂量PRP作用下细胞血小板源性生长因子(PDGF)的表达;荧光染色技术观察PRP作用下hDFbs在纯钛材料表面的生长;流式细胞术检测PRP培养后不同时间hDFbs细胞周期;CCK-8法检测不同浓度PRP培养条件下细胞增殖活力.结果:倒置相差显微镜下见PRP各浓度组细胞数量均多于对照组,细胞数量增加、折光性增强;免疫细胞化学检测,30 μL PRP组PDGF表达量最高且细胞密度最大,但10 μL PRP组累积吸光度值(IOD)高于20 μL PRP组(836.27±21.15 vs 794.35±30.26,P<0.05);荧光染色技术观察,PRP组材料表面hDFbs细胞密集,数量较对照组高;细胞周期检测,PRP促进细胞进入S期进行DNA复制,PRP作用后第2天PRP组S期细胞百分比高于空白组(34.41% vs 22.00%,P<0.05),第8天PRP组G0/G1期细胞百分比高于空白组(95.07% vs 89.70%,P<0.05);CCK-8测定细胞增殖活性,100.0%PRP组吸光度A450值高于12.5%PRP组(34.41% vs 22.00%,P<0.05).结论:高浓度的PRP虽然表现较强的促细胞增殖作用,但并不存在浓度、剂量依赖性,适宜浓度的PRP可促进hDFbs的增殖.     

海藻色素糖蛋白对小鼠H22肝癌细胞增殖与凋亡的影响

目的:研究麒麟菜海藻色素糖蛋白(seaweed pigment glycoprotein,SPG)对小鼠H22肝癌细胞增殖与凋亡的影响.方法:将不同浓度SPG与小鼠H22肝癌细胞共同培养,用MTT法测定癌细胞增殖活性.建立H22移植瘤小鼠肝癌模型,随机分为SPG高、中、低剂量组,对照组及环磷酰胺组.实验组小鼠每天分别给予不同剂量(100、50、10 mg/kg)的SPG灌胃,连续10 d,对照组同法给予等量生理盐水,环磷酰胺组小鼠腹腔注射20 mg/kg环磷酰胺,隔日一次.各组小鼠均于末次给受试物后24 h处死,取肿瘤组织用免疫组化法检测各组Bcl-2和Bax蛋白表达.结果:SPG高剂量组瘤细胞增殖活性(0.545±0.002)明显高于对照组(0.404±0.008)(P<0.05);SPG高剂量组Bcl-2和Bax蛋白阳性表达率分别为16.78%和38.1%,对照组分别为65.16%和4.68%,两组间的差异具有统计学意义(P<0.05).结论:SPG具有抑制小鼠H22肝癌细胞增殖、促进其凋亡的作用.     

葛根总黄酮诱导白血病细胞株凋亡及其分子机制的研究

目的 探讨葛根总黄酮(PR)对慢性粒细胞白血病(CML)细胞株K562和急性早幼粒细胞白血病(APL)细胞株NB4细胞增殖及凋亡的影响.方法 采用MTT法检测PR对K562细胞、NB4细胞的增殖抑制率;光学显微镜及荧光显微镜观察细胞形态改变;Hoechest33258荧光染色AnnexinV/PI双染法检测细胞凋亡率;DNA PI染色法分析细胞周期及亚二倍体峰.Western blot分别检测NB4细胞JNK、PARP、bcl-2、Caspase3,K562细胞bcr-abl、p53、bcl-2、Fas/FasL蛋白表达的变化.结果 12.5～200 μg/ml PR均能抑制K562、NB4细胞增殖.光学显微镜及荧光显微镜下观察到核固缩、凋亡小体等典型的细胞凋亡改变;Annexin V+/PI-细胞呈时间-剂量依赖性增加;DNA PI染色法发现细胞亚二倍体比例增加,G1期比例下降、S期比例增加.PR呈时间-剂量依赖性抑制K562细胞、NB4细胞增殖,诱导细胞凋亡.不同浓度PR干预后K562细胞bcr-abl蛋白水平呈浓度依赖性下调(F=18.74,P<0.05),而bcl-2则无明显变化;p53表达呈浓度依赖性上调;Fas/FasL表达无明显变化.NB4细胞JNK、PARP及Caspase 3蛋白表达与PR浓度呈正相关,与凋亡抑制蛋白bcl-2则呈负相关(F=42.32,P<0.05).结论 PR能有效抑制K562、NB4细胞增殖,阻滞细胞周期进程,诱导细胞凋亡,但分子机制不同.提示一定浓度PR具有较广谱的抗白血病效应.     

RNA干扰PTEN基因对HCT-8细胞增殖能力的影响

目的:观察小干扰RNA(siRNA)对结肠癌相关基因PTEN表达的抑制作用及其对结肠癌细胞增殖能力的影响.方法:构建携带有针对PTEN基因序列的siRNA真核绿色荧光表达载体重组质粒pGPU6/GFP/Neo-PTEN-1、pGPU6/GFP/Neo-PTEN-2、pGPU6/GFP/Neo-PTEN-3和pGPU6/GFP/Neo-PTEN-4,脂质体法转染入结肠癌HCT-8细胞(分别为p1、p2、p3和p4组),同时设转染阴性对照质粒组(p5组)和亲本细胞组(p6组),实时 PCR方法检测PTEN mRNA表达,MTT法分析细胞增殖活性变化.结果:通过荧光显微镜观察计数,细胞转染率为58%.实时 PCR方法检测结果显示,siRNA 重组质粒pGPU6/GFP/Neo-PTEN-1、pGPU6/GFP/Neo-PTEN-2、pGPU6/GFP/Neo-PTEN-3和pGPU6/GFP/Neo-PTEN-4的抑制率分别为48.3%、76.5%、29.4%和61.2%,与p6组比较差异均有统计学意义(P<0.05).MTT结果表明,细胞增殖抑制能力明显增强.结论:PTEN基因在肿瘤的发生发展中可能起到一定的作用.     

Taurolidine联合X射线对小鼠恶性黑色素瘤细胞周期进程的影响

目的:观察taurolidine联合X射线照射对小鼠恶性黑色素瘤(B16-4A5和B16-F10)细胞周期进程的影响,探讨其诱导肿瘤细胞凋亡的发生机制.方法:选择B16-4A5和B16-F10细胞系按给药浓度随机分为4组,taurolidine剂量分别为0、25、50和100 μmol·L-1,同时进行1、2和4 Gy X射线照射,采用流式细胞术检测细胞凋亡率和细胞周期,Western blotting分析cyclin B、cdc2和caspase-3的表达.结果:与25 μmol·L-1 taurolidine组比较,50和100 μmol·L-1 taurolidine组诱导B16-4A5和B16-F10细胞发生G0/G1期阻滞,细胞数分别升高54.9%、73.7%和36.8%、55.5%(P＜0.05); 50 μmol·L-1 taurolidine联合2和4 Gy X射线照射组,细胞G2/M期阻滞消除,细胞数分别降低52.1%、44.2%和59.3%、52.7%(P＜0.05).与对照组、单纯taurolidine组及单纯照射组比较,联合4 Gy X射线照射组cyclin B和cdc2的表达降低,caspase-3的表达升高(P＜0.05).结论:taurolidine联合X射线照射可去除G2/M期阻滞,可选择地抑制肿瘤细胞cyclin B和cdc2的表达、增强caspase-3的表达,共同诱导细胞凋亡.     

辛伐他汀联合阿糖胞苷对K562细胞增殖与凋亡的影响

目的 探讨辛伐他汀(SV)联合阿糖胞苷(Ara-C)对K562细胞增殖与凋亡的影响.方法 不同浓度SV和Ara-C单用或者联合处理K562细胞,对照组为K562细胞.药物作用24、48、72 h后收集细胞,分别观察各组细胞形态,采用MTT法检测不同组别细胞的生长抑制率,采用流式细胞术检测细胞早期凋亡率、细胞坏死比例.结果 SV联合Ara-C组与单药组相比细胞形态明显有核固缩现象,且可见凋亡小体形成,并且随着处理时间的增加,抑制率也增大.其中15 μmol/L SV联合20 μmol/LAra-C的细胞抑制作用最为显著,72 h细胞抑制率为(72±1)%,明显高于15 μmol/L SV组的(45±2)%和20μmol/LAra-C组的(44±0)%(P＜0.01),表现为协同抑制作用(24、48 h金氏Q值为1.24和1.19).流式细胞术检测发现20、15和10μmol/LSV组K562细胞早期凋亡率AnnexinV明显高于对照K562细胞(P＜0.01),而且随着时间延长和剂量的增大早期凋亡率也增加(P＜0.05).20和15 μmol/LSV组早期凋亡率均高于10 μmol/LSV组,而前两者之间差异无统计学意义(P＞0.05).晚期凋亡细胞率(PI)各组中差异均无统计学意义(P＞0.05).结论 SV体外抑制K562细胞增殖及诱导细胞凋亡,SV与Ara-C具有协同作用,增加了K562细胞对化疗药物的敏感性.15 μmol/L可能为SV体外最佳作用浓度.     

环巴胺诱导大肠癌Caco-2细胞凋亡效应和线粒体蛋白质组学研究

目的:探讨环巴胺对大肠癌Caco-2细胞的促凋亡效应,在线粒体蛋白水平阐明其作用机制.方法:取对数生长期大肠癌Caco-2细胞分为环巴胺处理组及阴性对照组,以5、10、20和40 μmol·L-1环巴胺处理Caco-2细胞,分别应用MTS法及流式细胞术测定Caco-2细胞生长抑制率及凋亡率,采用nano LC-ESI-MS方法检测环巴胺处理组与阴性对照组细胞线粒体蛋白质组学差异.结果:环巴胺处理组Caco-2细胞分别经10、20和40 μmol·L-1环巴胺作用24、48和72 h后,生长抑制率(分别为23.1%±1.8%,46.2±0.9%,53.4±2.3%;45.1%±2.8%,73.0%±2.5%,81.2%±1.6%;59.7±2.3%,87.5±1.4%,91±1.06%)明显高于阴性对照组(1.8%±0.2%,2.5%±0.1%,3.7%±0.3%)(P<0.01);经5、10、20和40 μmol·L-1环巴胺作用于Caco-2细胞48 h后,细胞凋亡率分别为24.1%±1.3%、31.7%±1.6%、50.5%±2.3%和64.0%±1.9%,明显高于阴性对照组(14.4%±0.7%)(P<0.01).蛋白质组学差异分析,环巴胺作用后,Caco-2细胞线粒体共有32种蛋白表达下调,25种蛋白表达上升,这些蛋白功能主要涉及细胞凋亡、细胞骨架、核酸、核糖体及蛋白质代谢等,其中与凋亡关系密切的蛋白包括ATF6、Clusterin、OPA1、RGD1565411和Cofilin.结论:环巴胺通过阻断Hedgehog(HH)通路,抑制大肠癌Caco-2细胞增殖,其机制与促进细胞凋亡有关;环巴胺可明显改变Caco-2细胞线粒体蛋白表达,其差异与凋亡关系密切.     

Comparison of lung dendritic cells and B cells in stimulating naive antigen-specific T cells

Dendritic cells (DCs) are specialized APCs that are important in priming naive T cells and can be manipulated in vitro and in vivo to enhance immunizations against microorganisms and tumors. A limitation in the development of suitable immunotherapeutic vaccines for the lung is incomplete information on the role of DCs and other potential APCs in the lung in priming naive T cells. In the current study, we analyzed the relative contributions of murine lung DCs and B cells to process and present OVA to naive CD4+ OVA323-339-specific (DO11.10) T cells in vitro. We also examined their expression of MHC class II and accessory molecules before and after maturation in culture. Similar to DCs from other sites, freshly isolated lung DCs can process OVA, spontaneously up-regulate MHC class II and accessory molecules during overnight culture, and stimulate naive T cells in an Ag-specific manner. In contrast, freshly isolated lung B cells were unable to both process and present native OVA. Furthermore, under conditions of limited OVA323-339 peptide exposure, B cells had a significantly diminished capacity to stimulate T cells, and this correlated with a decreased density of both MHC class II and important costimulatory molecules as compared with lung DCs.     

Ability of lymphokine-activated killer cells to lyse mycobacteria-infected cells

In cats and monkeys, we examined the parasympathetic component of the oculomotor complex, which directly innervates the ciliary muscle, using horseradish peroxidase (HRP). Labeled neurons of varying form and size were found in the Edinger-Westphal(EW) and the Perlia nuclei of the cat and in the anteromedian, EW, and Perlia nuclei of the monkey. Our study confirmed that a direct parasympathetic pathway exists from the midbrain to the ciliary muscles, and that accommodation is controlled in part by this direct link from the midsagittal region via a parasympathetic neuron of the oculomotor nuclear complex.     

Can cancer cells transform normal host cells into malignant cells?

A human prostate tumour cell line, LNCaP C4-2, when injected into athymic male nude mice, produced tumours containing: (1) only human cancer cells similar to those injected; (2) only murine stromal cells containing abnormal chromosome constitutions; or (3) both human prostate cancer cells similar to those injected and the transformed murine stromal cells with altered chromosome constitutions. Karyotypic analysis of murine metaphases from all the host-derived tumours showed mostly pseudodiploid chromosome constitutions, with multiple copies (amplification) of mouse chromosome 15 and the absence of a typical Y chromosome. Fluorescence in situ hybridization analysis of these murine cells, using a biotin-labelled total human DNA painting probe, further demonstrated the absence of human DNA and the presence of only mouse metaphase and interphase cells in these transformed stromal cells. These results suggest that cancer cells are capable of inducing neoplastic transformation in stromal cells of the host organ by some, as yet unknown, epigenetic mechanism(s).     

Differentiation of smooth muscle cells from undifferentiated cells of chicken gizzard occurs on the layer of fibroblast-like cells

Conflicting results have been reported in literature about the influence of beta-adrenergic stimulation on the fast cardiac sodium current (INa+). To elucidate these mechanisms in multicellular preparations we used the loose-patch-clamp technique to evaluate the effect of the beta-adrenergic agonist isoproterenol 1-1000 nmol/l. Isoproterenol enhanced INa+ at all membrane potentials by elevation of the maximal available INa+ . Only at the high concentration of 1 micromol/l was INa+ slightly depressed after depolarizing conditioning clamps. The most marked increase of the maximal available INa+ was 30+/-9% after application of 100 nmol/l isoproterenol. To learn about the mechanisms in view of sodium channel modulation we combined isoproterenol with the sodium channel blocker lidocaine (47 micromol/l). Under these circumstances the effects of both drugs were completely independent. This investigation shows clearly that low concentrations of isoproterenol increase INa+ in multicellular preparations by a gating-independent mechanism.     

Terminal differentiation of murine erythroleukemia cells: physical stabilization of end-stage cells

An important limitation in the use of the murine erythroleukenia (MEL) cell system as an in vitro system for the study of terminal erythroid differentiation has been the inability to produce significant numbers of cells which represent the end-point of the pathway in vitro. We show here that a major reason for the failure to observe end-stage cells in vitro is that such cells are physically unstable under the standard culture conditions used for MEL cell differentiation. Modification of these culture conditions by the addition of either bovine serum albumin or Ficoll leads to physical stabilization of end-stage cells. Under such culture conditions, uniform cultures of terminally differentiated MEL cells with morphological characteristics similar to those of normal mouse orthochromatophilic erythroblasts and reticulocytes are observed. Examination of physical and biochemical parameters of these cell populations give values which are similar to values characteristic of mouse reticulocytes. A physically stabilized MEL cell shows a narrow cell volume distribution with an average value of approximately 100 mum(3), similar to the cell volume distribution observed for mouse reticulocytes, while a typical MEL cell culture treated with DMSO but without a stabilizing agent exhibits a broader, more heterogeneous cell volume distribution with an average value of approximately 500 mum(3). Globin mRNA levels and levels of globin synthesis reach values almost equal to those in mouse reticulocytes in cultures of physically stabilized MEL cells while differentiating cultures not treated with a stabilizing agent reach substantially lower values for these parameters. We suggest that the ability to produce populations of MEL cells which undergo complete terminal erythroid differentiation in vitro will allow the analysis of the molecular mechanisms which control the terminal stages of the erythroid differentiation process.     

Artificial cells with emphasis on cell encapsulation of genetically engineered cells

Artificial cells are prepared in the laboratory for medical and biotechnological applications. Encapsulated cells are being studied for the treatment of diabetes, liver failure, and other conditions. More recently, there have been extensive studies into the use of encapsulated genetically engineered cells for gene therapy. We recently found that daily orally administered artificial cells, each containing a genetically engineered microorganism, can lower the elevated urea level in uremic rats to normal levels. This may solve the final obstacle of the lack of an effective oral urea removal system for the simple and inexpensive oral treatment of uremia. This is important because 85% of the world's uremic population cannot afford standard dialysis. Other areas of artificial cell application include use in hemoperfusion. Red blood cell substitutes based on modified hemoglobin are already in Phase 3 clinical trials in patients. Artificial cells containing enzymes are being developed for clinical trial in hereditary enzyme deficiency disease and other diseases. They are also being investigated for drug delivery and for use in other applications in biotechnology, chemical engineering, and medicine.     

Role of Langerhans cells and other dendritic cells in viral diseases

Langerhans cells are part of a vast system of potent antigen-presenting cells known under the name of dendritic cells. During the last decade, much has been learned on dendritic cell involvement in the immune response to infectious diseases. This review briefly summarizes our current understanding of the role played by Langerhans cells and other dendritic cells in the pathogenesis of DNA and RNA virus infections. These data may form the basis for the development of innovative approaches in the diagnosis, prevention, and treatment of viral diseases.     

Internalization of Chlamydia by dendritic cells and stimulation of Chlamydia-specific T cells

Chlamydia species are the causative agents of trachoma, various forms of pneumonia, and the most common sexually transmitted diseases. Although the infection cycle has been extensively characterized in epithelial cells, where the Chlamydia entry-vacuoles avoid fusion with host-cell lysosomes, the cellular immune response has received less attention. Moreover, despite the abundant presence of dendritic cells (DC) in the sites of infection, the interaction between Chlamydia and DC has never been studied. We observe that DC kill Chlamydia trachomatis and Chlamydia psittaci. The chlamydiae are internalized by the DC in a nonspecific manner through macropinocytosis, and the macropinosomes fuse subsequently with DC lysosomes expressing MHC class II molecules. The interaction induces maturation of the DC, since presentation of an exogenous Ag is severely inhibited after a 1-day incubation, although chlamydial Ags are still presented and recognized by Chlamydia-specific CD4+ T cells. Thus, DC most likely play a role in initiating the T cell response in vivo and could potentially be used in adoptive transfer therapies to vaccinate against Chlamydia.     

Human bone cells stimulate the growth of human breast carcinoma cells

Human breast cancer cells were cultured together with their metastatic target, bone tissue, to analyze possible growth promotion effects. The coculture of human osteosarcoma cells (TE-85) with human mammary carcinoma cells (ZR-75.1) resulted in up to 8.4-fold stimulation of proliferation of the breast tumor cells. Cell contact of the two cultures was permitted through the channels of Nuclepore filters. However, physical contact turned out not to be necessary, since the proliferative stimulus was also mediated by a bone-derived diffusible factor. Conditioned medium (CM), collected from human primary bone cultures, enhanced the rate of proliferation of several breast tissue cell lines (ZR-75.1, BT-20, HBL-100), while some lines were not affected by osteoblast CM. Breast tissue lines responding to bone CM express low to intermediate levels of the c-erbB-2 gene, in contrast to nonstimulated lines, which overexpress the gene. Recent observations of metastatic spread in breast cancer patients suggest a distinctive pattern of secondary tumor distribution in association with c-erbB-2 protein expression. Bone tissue seems to be a preferential target for metastases of c-erbB-2-negative breast tumors.