砷化合物致人表皮癌A431细胞的毒性及氧化应激和砷代谢研究

目的探讨不同砷化合物致人表皮癌细胞(A431)的细胞毒性及氧化应激和砷代谢的情况。方法培养的A431细胞分别暴露于0.05～50.0μmol/L一甲基亚胂酸(MMAⅢ),0.05～200.0μmol/L三氧化二砷(As2O3,As3+),0.5～500.0μmol/L砷酸氢二纳(As5+)和二甲基胂酸钠(DMAⅤ)24 h,应用四甲基偶氮唑盐(MTT)法测定细胞生存率;检测砷化物对丙二醛(MDA)含量、超氧化物歧化酶(SOD)活力的影响;以流式细胞仪检测细胞内活性氧(ROS);以原子荧光分析方法检测细胞内外不同形态砷含量。结果在一定剂量范围的MMAⅢ(≥5.0μmol/L)、As2O(3≥200.0μmol/L)、砷酸氢二钠(0.5,≥200.0μmol/L)和二甲基胂酸钠(500.0μmol/L)能够显著降低A431的细胞生存率(P<0.05,P<0.01),而在低剂量时,四种砷化物能显著促进A431细胞增殖(P<0.05,P<0.01)。与对照组相比,50.0μmol/L MMAⅢ组,50.0、250.0μmol/LAs2O3组,0.5μmol/L砷酸氢二钠组MDA含量均显著升高(P<0.05,P<0.01),而250.0μmol/L砷酸氢二钠和5.0～250.0μmol/L二甲基胂酸钠组MDA含量均显著降低(P<0.05,P<0.01)。与对照组相比,0.5、50.0μmol/L MMAⅢ组,5.0～250.0μmol/L砷酸氢二钠组和0.5～500.0μmol/L二甲基胂酸钠组的SOD活力显著升高(P<0.05,P<0.01)。细胞内活性氧含量依次为0.5μmol/L MMAⅢ>50.0μmol/L As2O3>100.0μmol/L砷酸氢二钠>100.0μmol/L二甲基胂酸钠。低剂量As2O3、砷酸氢二钠组细胞内砷甲基化率高于高剂量组,且As2O3组甲基化率高于砷酸氢二钠。结论在本实验条件下,不同砷化物在低剂量促进A431细胞增殖,高剂量抑制增殖,可能与A431细胞的氧化应激和砷代谢有关。     

顺铂诱导人宫颈癌Hela细胞凋亡及其对细胞周期影响的研究

目的:观察顺铂对人宫颈癌Hela细胞凋亡及细胞周期的影响,以揭示顺铂治疗宫颈癌的机理。方法:运用体外细胞培养技术,以不同浓度的顺铂作用于培养的人Hela细胞72 h,用流式细胞仪分析其凋亡率及细胞周期。结果:顺铂能以浓度依赖的方式诱导Hela细胞凋亡,并呈现出G0/G1期阻滞作用。结论:顺铂能诱导Hela细胞凋亡并改变其细胞周期分布,阻滞Hela细胞于G0/G1期,这可能是顺铂抗宫颈癌作用的重要机制之一。     

miR-214对HepG2细胞增殖及细胞周期影响

目的 探讨miR-214对HepG2细胞增殖及细胞周期的影响。方法 Realtime-PCR法检测肝癌细胞株SMMC-7721、HepG2、SK-Hep-1、Huh 7、Hep3B及正常肝细胞L-02中miR-214表达量,并利用脂质体转染miR-214 NC及miR-214 mimics,采用Realtime-PCR法检测转染效果;采用MTT法、流式细胞术分别检测miR-214对肝癌细胞活力、凋亡及细胞周期影响;蛋白印迹（WB）检测miR-214对肝癌细胞中细胞周期蛋白D1（cyclinD1）,细胞周期蛋白依赖性激酶4（CDK4）,生存素（survivin）及增值细胞核抗原（PCNA）表达影响。结果 miR-214在肝癌细胞SMMC-7721、SK-Hep-1、Huh 7、Hep3B、HepG2中表达量[分别为（1.25±0.10）、（1.43±0.10）、（0.95±0.09）、（0.98±0.01）、（0.82±0.08）]明显低于正常肝细胞L-02（2.52±0.23）,其中HepG2中miR-214表达量最低。miR-214 mimics组HepG2细胞中miR-214表达量（2.38±0.23）明显高于miR-214 NC组（0.83±0.08）,miR-214 mimics组HepG2细胞活力（0.37±0.03）明显低于miR-214 NC组（0.78±0.07）;与miR-214 NC组比较,miR-214 mimics组HepG2细胞凋亡率明显升高,细胞周期阻滞于G1期,cyclinD1、CDK4、survivin及PCNA表达量下调,差异均有统计学意义（P<0.01）。结论 上调miR-214表达可抑制肝癌细胞增殖,其机制可能与调控细胞周期相关蛋白表达水平有关。     

Differential effect of CD4Foxp3 T-regulatory cells on the B and T helper cell responses to influenza virus vaccination

The T-regulatory (T-reg) cells restrict the T-cell functions in various viral infections including influenza infection. However little is known about the effect of T-regs in influenza vaccination. Herein, we found that immunization of BALB/c mice with a prototype of UV-inactivated influenza PR8/A/34 virus vaccine expanded the CD4⁺Foxp3⁺ T-reg pool and fostered the development of virus-specific CD4⁺Foxp3⁺ T-reg cells. Increasing the size of Foxp3⁺ T-reg pool did not alter the primary PR8-specific B-cell response, but it did suppress the primary and memory PR8-specific T helper responses induced by vaccination. In contrast, the vaccination-induced T helper cell response was augmented in the absence of CD4⁺Foxp3⁺ T-reg cells. Since CD4 T helper cells contribute to anti-influenza protection, therapeutic “quenching” of T-reg function prior to vaccination may enhance the efficacy of influenza vaccination.     

叶绿酸对恶性转化细胞周期及体外生长的影响

目的研究叶绿酸对反式7，8二羟9，10环氧苯并(a)芘(反式BPDE)恶性转化人支气管上皮细胞系(16HBE)体外生长及细胞周期影响。方法采用噻唑蓝(MTT)比色法和流式细胞术(FCM)，同时观察正常人支气管上皮细胞(16HBE)、反式-BPDE恶性转化细胞系、叶绿酸抗反式-BPDE恶性转化细胞系生长曲线和生长周期的变化以及经叶绿酸处理后的影响。结果BPDE恶性转化细胞系增殖活性明显大于正常细胞系(16HBE)，经100μmol／L叶绿酸抗BPDE恶性转化细胞组增殖活性较转化组有一定降低。经同样浓度叶绿酸处理的正常细胞组，未见降低细胞增殖活性作用。反式-BPDE恶性转化细胞组G0／G1期细胞周期比例明显少于正常组细胞。但经叶绿酸抗转化组G0／G1期比例有所提高，而经同样浓度叶绿酸处理的正常细胞组G0／G1期细胞周期比例无明显改变。结论对正常细胞无影响的一定浓度叶绿酸有抑制反式-BPDE恶性转化人支气管上皮细胞增殖活性的作用，并通过提高恶性转化细胞G0／G1期细胞周期比例而影响细胞周期的进程，从而改变恶性细胞的增殖和分化。     

1，25-二羟基维生素D3对K562细胞周期及凋亡的影响

目的观察1，25-二羟基维生素D3[1，25（OH）2D3]对白血病细胞株K562细胞周期及细胞凋亡的作用。方法Western印迹检测维生素D受体（VDR）在K562细胞的表达；四噻唑蓝法（MTT）、AO／EB、流式细胞仪分析细胞生长抑制率、细胞凋亡率及细胞周期。结果（1）K562细胞核阳性表达VDR；（2）0-10^-6mol／L浓度的1，25（OH）2D3呈浓度依赖性抑制K562细胞增殖，10^-8mol／L 1，25（OH）2D3明显抑制K562细胞增殖，促进凋亡，细胞周期阻滞主要发生在G2期或M早期，凋亡率从4．1％（对照组）增至26．5％（P〈0．01）。结论1，25（OH）2D3可明显抑制K562细胞增殖，促进细胞凋亡。     

UVC-induced apoptosis in human epithelial tumor A431 cells: sequence of apoptotic changes and involvement of caspase (-8 and -3) cascade

Human epidermoid tumor A431 cells underwent apoptosis following exposure to ultraviolet C (UVC). The apoptosis was of the interphase death type, and mostly occurred within one cell cycle, independent of the cell-cycle phases. We further examined the detailed sequential order of apoptotic changes in cells after UVC exposure and the involvement of caspases using six caspase inhibitors. The loss of mitochondrial transmembrane potential (delta psi m) appeared in the earliest phase; subsequently, the chromatin condensation and DNA-fragmentation occurred. Cell shrinkage and loss of the plasma-membrane integrity, judged by propidium iodide (PI) staining, were observed in the later phase. A broad-spectrum caspase inhibitor, z-VAD-fmk, completely prevented all apoptotic changes, except for the depletion of delta psi m. Both Ac-DEVD-CHO and Ac-IETD-CHO, inhibitors of caspase -3 and -8, respectively, effectively inhibited typical chromatin condensation to almost the same extent. However, the nuclei still showed partial condensation. A caspase -9 inhibitor, Ac-LEHD-CHO, did not prevent chromatin condensation, though it partially inhibited cell-size reduction and PI-stainability. None of the caspase inhibitors could inhibit the delta psi m reduction. These results strongly suggest that the collapse of delta psi m is not a part of the central apoptotic machinery, and that caspase cascade(s), especially caspase-8 to -3, play an important role in UVC-induced apoptosis in A431.     

A West Nile virus NS4B-P38G mutant strain induces cell intrinsic innate cytokine responses in human monocytic and macrophage cells

Previous studies have shown that an attenuated West Nile virus (WNV) nonstructural (NS) 4B-P38G mutant induces stronger innate and adaptive immune responses than wild-type WNV in mice, which has important applications to vaccine development. To investigate the mechanism of immunogenicity, we characterized WNV NS4B-P38G mutant infection in two human cell lines—THP-1 cells and THP-1 macrophages. Although the NS4B-P38G mutant produced more viral RNA than the parental WNV NY99 in both cell types, there was no detectable infectious virus in the supernatant of either cell type. Nonetheless, the attenuated mutant boosted higher innate cytokine responses than virulent parental WNV NY99 in these cells. The NS4B-P38G mutant infection of THP-1 cells led to more diverse and robust innate cytokine responses than that seen in THP-1 macrophages, which were mediated by toll-like receptor (TLR)7 and retinoic acid-inducible gene 1(RIG-I) signaling pathways. Overall, these results suggest that a defective viral life cycle during NS4B-P38G mutant infection in human monocytic and macrophage cells leads to more potent cell intrinsic innate cytokine responses.     

Investigation of TF-binding lectins from dietary sources and SRL on proliferation and cell cycle progression in human colon HT29 and SW620 cells

TF antigen binding lectins from dietary sources PNA, ACA, ABL, JAC, and SRL from Sclerotium rolfsii have been reported to induce diverse effects on cancer cell proliferation by different mechanisms. This study aimed to compare effects of these lectins on growth and cell cycle progression in colon cancer HT29 and SW620 cells. As reported SRL, ABL, and JAC inhibited while PNA and ACA increased cell proliferation. ABL and JAC treated HT29 cells showed increased cell population in G0/G1 phase. PNA, ACA, ABL, and JAC increased SW620 cell population in S and decreased in G2/M phase. In contrast, SRL and JAC increased hypodiploid population in both the cells. PNA and ACA reduced whereas SRL and ABL diminished cell cyclin D1 expression. SRL, PNA, and ACA also reduced cellular cyclin D3 level while SRL, ABL, and JAC reduced cyclin E levels. ABL decreased CDK5 levels while SRL and ACA completely abolished CDK5 expression. All the lectins completely abolished cyclin D2 expression. These results not only confirms growth regulatory effects of TF-binding lectins but also indicates different effects of these lectins on cell growth is associated with regulation on expression of cell cycle associated proteins in G1-S phase and on cell cycle progression.     

Inhibitory effect of liquiritigenin on migration via downregulation proMMP-2 and PI3K/Akt signaling pathway in human lung adenocarcinoma A549 cells

Liquiritigenin (LQ) is a flavanone extracted from Glycyrrhizae, which has multiple biological effects, such as antiinflammation and anticancer. This study is the first to investigate the effect of LQ on the migration of human lung adenocarcinoma A549 cells in vitro. First, LQ exhibited inhibitory effects on the adhesion and migration of A549 cells in the absence of cytotoxicity. Gelatin zymography and Western blot analysis showed that LQ significantly reduced the expression of promatrix metalloproteinase-2 (proMMP-2) in A549 cells in terms of both activity and protein level. Second, LQ inhibited the phosphorylation of Akt and activated the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Furthermore, the treatment of inhibitors specific for Akt (LY294002) and ERK1/2 (U0126) to A549 cells resulted in reduced activity of proMMP-2. These results suggested that the inhibition on proMMP-2 expression by LQ may be through suppression on PI3K/Akt signaling pathway, which in turn led to the inhibition of lung adenocarcinoma A549 cells migration. However, activation of ERK might not be involved in the regulation of proMMP-2. Taken together, LQ may be considered as a potential interfering agent of cancer progression.     

Synergistic effect of heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin and X-rays, but not carbon-ion beams, on lethality in human oral squamous cell carcinoma cells

The purpose of this study is to clarify the effect of a heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in combination with X-rays or carbon-ion beams on cell killing in human oral squamous cell carcinoma LMF4 cells. Cell survival was measured by colony formation assay. Cell-cycle distribution was analyzed by flow cytometry. Expression of DNA repair-related proteins was investigated by western blotting. The results showed 17-AAG to have synergistic effects on cell lethality with X-rays, but not with carbon-ion beams. The 17-AAG decreased G(2)/M arrest induced by X-rays, but not by carbon-ion beams. Both X-ray and carbon-ion irradiation up-regulated expression of non-homologous end-joining-associated proteins, Ku70 and Ku80, but 17-AAG inhibited only X-ray-induced up-regulation of these proteins. These results show that 17-AAG with X-rays releases G(2)/M phase arrest; cells carrying misrepaired DNA damage then move on to the G(1) phase. We demonstrate, for the first time, that the radiosensitization effect of 17-AAG is not seen with carbon-ion beams because 17-AAG does not affect these changes.     

米非司酮对绒毛组织中MTA3、ER及PR的影响

目的:了解米非司酮对绒毛组织中MTA3(Metastasis-associatedgene3)的表达及对雌、孕激素受体的影响。方法:对120例停经49天以内临床证实为早期妊娠,要求终止妊娠的妇女,根据停经天数的不同分为停经45天以下及45～49天两大组,每组60例,各组随机分为3小组,分别予米非司酮100mg,200mg,以及对照组。应用RT-PCR法检测各组MTA3的表达。并从各组中各自随机挑选5例,用ERα、PR单克隆抗体免疫组织化学法检测绒毛组织的ERα、PR水平。结果:4组用米非司酮的早孕妇女绒毛中MTA3表达均较对照组增高,差异有统计学意义(P<0.05),并表现为剂量相关性,随着米非司酮的剂量增加,MTA3的表达增高。服药组ERα水平增高,阳性物质定位于胞浆中,胞核中阴性。服药组PR水平低于对照组。结论:米非司酮用于早孕妇女可能通过直接或间接的途径影响雌激素通路,导致滋养细胞m-RNA水平MTA3表达增多,从而有可能使一些细胞黏附分子丢失而导致滋养细胞的侵袭能力的改变,达到抗早孕目的。     

Cellular toxicity in Chinese hamster ovary cell cultures. II: A statistical appraisal of sensitivity with the rabbit alveolar macrophage,Syrian hamster embryo,BALB 3T3 mouse,and human neonatal fibroblast cell systems

Chinese hamster ovary, rabbit alveolar macrophage, Syrian hamster embryo, BALB 3T3 mouse, and human neonatal fibroblast cells were employed in a statistical evaluation of the relative sensitivity of the cells to toxic substances. The cells were exposed to 1,2,4-trichlorobenzene, 2,4-dimethylphenol, Aroclor 1248, cadmium chloride, lead sulfate, nickel nitrate, lead oxide-coated fly ash, and a fine particulate from coal combustion. A filter-disk technique was used to measure the inhibition of protein and DNA synthesis. A quantitative ranking of cell-system sensitivity was determined from comparisons of statistically significant differences (P ? 0.01) in protein and DNA synthesis expressed as a percentage of control. An overall ranking of sensitivity showed that rabbit alveolar macrophages, Syrian hamster embryo cells, and Chinese hamster ovary cells were more sensitive than another of the five cell systems in 75, 68, and 62% of the experiments, respectively. The corresponding values for BALB 3T3 mouse and human neonatal fibroblast cells were 38 and 28%, respectively, under our experimental conditions. Detailed data on the control cell cultures are also presented.     

Expression of human blood group antigens A and B in stomach cells of C. carpio, C. auratus, R. ridibunda and H. sapiens

Human blood group ABH antigens are found not only on red blood cell membranes, but in many other cell types as well. Their biological functions still remain unclear. The aim of the present study was to examine the cellular expression of these antigens in the stomach of representatives of different Vertebrates--Pisces and Amphibia and to compare it with their expression found in Man. The immunohistochemical technique applied was based on the biotinstreptavidin-peroxidase complex. Monoclonal antibodies to human A and B antigens were used as primary antibodies in the system. Stomach paraffin sections from Cyprinus carpio, Carassius auratus, Rana ridibunda and Homo sapiens were examined. Blood group antigens were found mainly in tunica mucosa of C. carpio, C. auratus and R. ridibunda. Tunica muscularis and tunica serosa were always immunonegative. The antigens were localized in the apical part of the cytoplasm of epithelial cells in lamina epithelialis. Strong positive reaction was seen in secretory granules of the stomach of C. auratus, while in R. ridibunda the antigens were expressed by single epithelial cells in cardial stomach glands. A and B antigens were not found in human stomach sections most probably due to the negative secretor status of the individuals studied. Our results show that ABH human blood group antigens are evolutionary conserved structures similarly expressed by different Vertebrates. The phylogenetic stability in their cellular expression possibly results from the important biological role they have. Future large scale systematic investigations could elucidate the undefined and disputable physiological functions of human blood group antigens.     

Efficient synthesis of 6-(hetero)arylthieno[3,2-b]pyridines by Suzuki-Miyaura coupling. Evaluation of growth inhibition on human tumor cell lines, SARs and effects on the cell cycle

A wide variety of new bi(hetero)aryl derivatives of the thieno[3,2-b]pyridine skeleton was obtained in high to excellent yields (65-91%) by Suzuki-Miyaura cross-coupling of the methyl 3-amino-6-bromothieno[3,2-b]pyridine-2-carboxylate, recently reported by us, with aryl or heteroaryl pinacolboranes or potassium trifluoroborates. The coupling products obtained were evaluated for their growth inhibitory effect on three human tumor cell lines, representing different tumor models, MCF-7 (breast adenocarcinoma), A375-C5 (melanoma) and NCI-H460 (non-small cell lung cancer). Some of the compounds showed an interesting activity against the tested cell lines, with GI50 values in the μM range, and it was possible to establish some structure-activity relationships (SARs). Several compounds presented GI50 values below 15 μM, particularly a bithiophene and an o-aniline thienopyridine derivative. The first presented selectivity for MCF-7 and NCI-H460 cell lines, with very low GI50 values (0.7-1.0 μM), while the latter was active against the three cell lines tested in this study, also presenting very low GI50 values (2.5-4.2 μM). The effect of these two compounds on cell cycle progression was analyzed in the NCI-H460 cell line. Results showed that both compounds interfered with the normal cell cycle distribution.