The effect ofin vitro andin vivo dexamethasone on human neutrophil function

There is a significant fall in PMN chemotaxis to the peptide FMLP in response to increasing concentrations of dexamethasonein vitro. The response fell in a dose related manner from a control value of 53.7 SE±9.6 cells per high power field (cpf) to 47.3 SE±8.1 at 10^–6 M (p<0.05) and 24.7±8.9 at 10^–3 M (p<0.025). A similar response was observed for the chemoattractants zymosan activated serum and the sol phase of purulent sputum. The effect was independent of protein synthesis or the period of incubation. Twelve milligrams of dexamethasone taken daily by 6 healthy volunteers resulted in a significant (p<0.025) reduction in the chemotactic response of PMN to 10^–8 M FMLP (from 29.5±1.55 to 13.7±1.8 cpf) which was apparent within 2 hours of taking the first dose. This effect was sustained for the three days on which dexamethasone was taken but returned to normal 7 days after the last dose had been administered.Dexamethasone therapy had no effect on unstimulated PMN superoxide anion production eitherin vitro orin vivo.Thein vivo effect on neutrophil function occurred at mean serum dexamethasone concentrations of 1.26 (±0.28)×10^–7 M on day 1, 1.44 (±0.15)×10^–7 M on day 2 and 1.31 (±0.13)×10^–7 M on day 3. Thus we conclude that dexamethasone concentration which inhibit PMN chemotaxisin vivo are much lower than those required to exert the same effectin vitro.     

Effect of dexamethasone and ACC on bacteria-induced mucin expression in human airway mucosa

Gram-negative bacteria can stimulate mucin production, but excessive mucus supports bacterial infection and consequently leads to airway obstruction. Therefore, the effect of dexamethasone (DEX) and the antioxidant acetyl-cysteine (ACC) on bacteria-induced mucus expression was investigated. Explanted human airway mucosa and mucoepidermoid cells (Calu-3) were stimulated with lipopolysaccharide (LPS) or PAM3 (a synthetic lipoprotein). DEX or ACC were added to either LPS- or PAM3-stimulated airway mucosa or Calu-3 cells. Mucin mRNA expression (MUC5AC) and total mucus glycoconjugates (mucin protein) were quantified using real-time PCR and periodic acid Schiff staining. LPS and PAM3 significantly increased mucin expression in airway mucosa and Calu-3 cells (P < 0.05). DEX alone had no significant effect on mucin expression in airway mucosa or Calu-3 cells (P > 0.05). In contrast, DEX significantly reduced LPS- and PAM3-induced mucin expression in explanted mucosal tissue and mucin expression in Calu-3 cells (P < 0.05). In explanted human airway mucosa ACC alone significantly increased mucin expression (P < 0.05). In contrast, ACC significantly decreased LPS- and PAM3-induced mucin expression (P < 0.05). In Calu-3 cells ACC alone had no significant effect on mucin expression (P > 0.05). ACC decreased LPS- and PAM3-induced mucin expression, but this effect was not significant (P > 0.05). These data suggest that DEX can effectively reduce bacteria-induced mucin expression in the airways. ACC alone may increase mucin expression in noninfected mucosa, but it decreased bacteria-induced mucin expression. Further studies are warranted to evaluate whether the effect of DEX or ACC is clinically relevant.     

Histone H1 expression in human prostate cancer tissues and cell lines

Histone H1, one of the histone superfamilies, is known to determine chromatin structure and alter gene expression. It also contributes to regulation of cell proliferation in breast cancer. We hypothesized a similar association in prostate cancer, and therefore examined relationships between histone H1 expression and Gleason pattern, Ki-67 and androgen receptor levels in a series of prostate cancer tissues and cell lines. Histone H1 positive cancer cells increased with the Gleason pattern. Gleason pattern 3 tumors were divided into two groups, one with high histone H1 positivity (H1-high cases, 60-100% positivity) and the other with low histone H1 positivity (H1-low cases, 0-20% positivity). Ki-67 or androgen receptor positivity in H1-high cases was significantly higher than in H1-low cases. PC3 cells demonstrated more frequent histone H1 and Ki-67 positivity as compared to LNCaP cells. Silencing of histone H1 by siRNA transfection significantly reduced cell proliferation in LNCaP and PC3. These findings suggest that histone H1 expression is associated with the Gleason pattern, cell proliferation and androgen receptor expression in prostate cancers.     

Gene and microRNA expression reveals sensitivity to paclitaxel in laryngeal cancer cell line

Paclitaxel is a widely used chemotherapy drug for advanced laryngeal cancer patients. However, the fact that there are 20-40% of advanced laryngeal cancer patients do not response to paclitaxel makes it necessary to figure out potential biomarkers for paclitaxel sensitivity prediction. In this work, Hep2, a laryngeal cancer cell line, untreated or treated with lower dose of paclitaxel for 24 h, was applied to DNA microarray chips for gene and miR expression profile analysis. Expression of eight genes altered significantly following paclitaxel treatment, which was further validated by quantitative real-time PCR. Four up-regulated genes were ID2, BMP4, CCL4 and ACTG2, in which ID2 and BMP4 were implicated to be involved in several drugs sensitivity. While the down-regulated four genes, MAPK4, FASN, INSIG1 and SCD, were mainly linked to the endoplasmic reticulum and fatty acid biosynthesis, these two cell processes that are associated with drug sensitivity by increasing evidences. After paclitaxel treatment, expression of 49 miRs was significantly altered. Within these miRs, the most markedly expression-changed were miR-31-star, miR-1264, miR-3150b-5p and miR-210. While the miRs putatively modulated the mRNA expression of the most significantly expression-altered genes were miR-1264, miR-130a, miR-27b, miR-195, miR-1291, miR-214, miR-1277 and miR-1265, which were obtained by miR target prediction and miRNA target correlation. Collectively, our study might provide potential biomarkers for paclitaxel sensitivity prediction and drug resistance targets in laryngeal cancer patients.     

川陈皮素对非小细胞肺癌A549细胞的抑制作用

目的:观察川陈皮素(5,6,7,8,3′4′-hexamethoxyflavone)对非小细胞肺癌A549细胞的抑制作用并研究其作用机制。方法:以不同浓度的川陈皮素作用于A549细胞,分别用MTT法、细胞生长曲线研究川陈皮素对A549的增殖抑制作用,Giemsa染色观察细胞形态变化。结果:MTT提示川陈皮素浓度为4mg·L-1~128mg·L-1时,对A549的48、72小时抑制率为15%~85%,48小时IC50为25.7mg·L-1,72小时IC50为16.7mg·L-1;生长曲线提示川陈皮素对A549细胞的抑制作用呈明显时效和量效关系;Giemsa染色后细胞出现明显的凋亡形态学变化。结论:川陈皮素对非小细胞肺癌A549细胞具有增殖抑制作用,其机制可能是诱导细胞凋亡。     

双氢青蒿素诱导人前列腺癌细胞系PC-3凋亡

目的探讨双氢青蒿素(dihydroartemisinin,DHA)对人前列腺癌细胞系PC-3的凋亡诱导作用,并探讨其可能机制。方法人前列腺癌PC-3细胞经不同浓度(0、25、50和100μmol/L)DHA处理48 h,用FCM法检测各组细胞凋亡率。用荧光定量PCR检测细胞中HSP70 mRNA的表达。用蛋白质印迹法检测细胞中HSP70蛋白、凋亡酶激活因子(Apaf-1)及caspase-3的表达;荧光定量PCR及蛋白质印迹法增加两组,即100μmol/L HSP70抑制剂槲皮素(quercetin)作为阳性药物对照组,以DMSO作为溶剂对照组。结果 DHA能明显诱导PC-3细胞凋亡(P0.05)。不同浓度DHA能明显下调HSP70 mRNA及蛋白表达水平(P0.05),上调Apaf-1及caspase-3蛋白表达水平(P0.05)。结论双氢青蒿素能诱导前列腺癌PC-3细胞凋亡,其作用机制可能是DHA干扰HSP70的表达,促进caspase信号通路中Apaf-1及caspase-3表达。     

乙型肝炎病毒在体内外抑制补体C3和C4表达的研究

目的 探讨乙型肝炎病毒(HBV)对补体C3和C4表达的影响,并探讨其调节机制.方法 采用基因芯片筛选HepG2和HepG2.2.15细胞的差异表达基因,免疫比浊法检测HBV患者和健康对照者补体C3和C4血清学水平,将HBV感染性克隆pHBV1.3转染HepG2细胞,RT-PCR和Western blot法检测C3和C4表达水平的变化.结果 补体C3和C4 mRNA在HepG2.2.15细胞中水平降低;C3和C4在慢性乙型肝炎患者和肝癌患者的血清学水平明显低于健康对照者(P＜0.05);HBV能够在mRNA和蛋白水平下调C3和C4的表达.结论 HBV能够在体内外抑制补体C3和C4的表达.     

Proinflammatory cytokines trigger MUC gene expression and mucin release in the intestinal cancer cell line LS180

OBJECTIVES AND DESIGN: Proinflammatory cytokines and a defective mucus layer are involved in the pathogenesis of colitis. Therefore, we determined cytokine effects on MUC gene expression and mucin secretion. MATERIALS AND METHODS: LS180 cells were characterized by light and electron microscopy and subsequently exposed to interleukin 1 (IL-1, 1 ng/ml), interleukin 6 (IL-6, 10 ng/ml), or tumor necrosis factor-alpha (TNFalpha, 10 ng/ml). MUC gene (MUC2, MUC5AC, MUC5B, MUC6) mRNA expression was assessed by RT-PCR, the encoded proteins were identified by immunocytochemistry and Western blotting, and the released mucins were isolated and chromatographically characterized. RESULTS: Thirty to 40% of the cells contained intracellular mucin granules. Incubation with IL-1 transiently stimulated the mRNA expression of MUC2 and MUC5AC, whereas IL-6 induced an early response of MUC2, MUC5B and MUC6. TNFalpha upregulated the expression of MUC2 and MUC5B for 3 hours, and had no effect on the expression of MUC 5AC and MUC6. Immunocytochemistry and Western blotting confirmed TNFalpha effects on MUC2 and MUC5AC on the protein levels. All cytokines stimulated the release of less glycosylated mucins and considerably modulated their carbohydrate composition. CONCLUSION: Our data demonstrate differential cytokine effects on mucin synthesis, secretion and composition. These alterations may contribute to the defective mucus layer in colitis.     

促性腺激素释放激素类似物对人乳腺癌细胞的影响

目的:探讨促性腺激素释放激素(gonadotropin-releasing hormone,GnRH)类似物曲普瑞林(triptorelin)对人乳腺癌细胞株MCF-7、MDA-MB-231细胞生长及细胞外信号调节蛋白激酶(extracellular signal-regulated kinase,ERK)/丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)和磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)信号通路中重要信号分子ERK1/2和Akt活化的影响。方法:使用不同浓度、不同时间的曲普瑞林刺激人乳腺癌MCF-7及MDA-MB-231细胞株,MTT法检测细胞增殖,流式细胞术检测细胞周期的分布,Western blotting检测Akt和ERK1/2的磷酸化程度。结果:曲普瑞林(10-5mol/L)作用人乳腺癌MCF-7细胞192 h、曲普瑞林(10-4mol/L)作用人乳腺癌MCF-7细胞168 h、192 h或曲普瑞林(10-4mol/L)作用人乳腺癌MDA-MB-231细胞192 h可明显抑制细胞生长(P0.05);曲普瑞林(10-4mol/L)作用人乳腺癌MCF-7和MDA-MB-231细胞192 h,ERK1/2的磷酸化程度均较正常对照组低,Akt的磷酸化程度较正常对照组高,但差异无统计学意义(P0.05)。结论:GnRH类似物曲普瑞林对人乳腺癌细胞的抑制作用,不仅仅是通过对垂体激素的降调节机制,还可能产生直接抑制作用。但该抑制作用未涉及ERK/MAPK和PI3K/Akt信号通路。     

LASP-1稳定转染对人结肠癌细胞株SW480细胞增殖及成瘤能力的影响

目的 探讨LIM和SH3蛋白1(LASP-1)对人结直肠癌细胞株体内外增殖能力的影响.方法 用逆转录聚合酶链反应(RT-PCR)和Western blot方法检测LASP-1在结直肠癌细胞株中的表达,筛选LASP-1不/低表达细胞株;将LASP-1 cDNA导入内源性低表达LASP-1基因的人结直肠癌细胞株SW480细胞中,同时构建带绿色荧光蛋白(CFP)的表达载体转染SW480细胞株,G418筛选抗性克降,经鉴定后利用四甲基偶氮唑盐(MTT)法检测LASP-1对细胞体外增殖的影响,通过整体成像系统观察LASP-1对细胞在裸鼠体内成瘤能力及肿瘤生长能力的影响.结果 成功构建pcDNA3-LASP-1和pEGFP-LASP-1表达载体,将其稳定转染低表达LASP-1的SW480细胞中.通过7 d连续比较,LASP-1基因的导入明显促进结直肠癌细胞的体外增殖能力.裸鼠皮下注射携带GFP的稳定细胞系,整体成像观察过表达LASP-1的细胞成瘤能力和肿瘤生长速度均强于对照细胞.结论 LASP-1基因具有促进结直肠癌细胞增殖的能力,可能作为结直肠癌发生过程中一个有价值的指标.

Abstract：

Objective To investigate the effect of LIM and SH3 protein 1 (LASP-1)expression on the proliferative ability of human colorectal cancer cells in vitro and in vivo. Methods RT-PCR and Western blot were used to screen cells of the colorectal cancer cell line with no or with minimal endogenous LASP-1 expression. LASP-1 cDNA with or without GFP was transfected into SW480 colorectal cancer cells with minimal LASP-1 expression. Stable transfectants were established after G418 selection. Cell proliferative capacity was assessed by MTT assay. Tumor growth was visualized by whole-body imaging system. Results pcDNA3-LASP-1 and pEGFP-LASP-1 vectors were successfully constructed and transfected into the SW480 cells. After comparative study for 7 days, LASP-1 over-expression was found capable of enhancing signific antly the proliferation of colorectal cancer cells in vitro. Stable transfectants with GFP expression were inoculated subcutaneously into the nude mice. Tumorigenesis and proliferation ability of LASP-1-overexpressed transfectants were higher than those of the control cells. Conclusion LASP-1 gene expression enhances proliferation of colorectal cancer cells and may serve as a useful marker for colorectal cancer progression.     

CD3AK细胞对人卵巢癌细胞系的体外杀伤作用

目的：研究卵巢癌盆腔引流淋巴结的淋巴细胞,经ＣＤ３单克隆抗体激活后的杀伤细胞ＣＤ３ＡＫ（ＣＤ３ａｃｔｉｖａｔｅｄｋｉｌｅｒｃｅｌｓ）在体外的抗瘤作用。方法：采用卵巢癌病人盆腔引流淋巴结的淋巴细胞,在体外与ＣＤ３单抗和少量ｒＩＬ－２诱导并激活后,在两株人卵巢癌ＨＯ－８９１０、ＨＯ－８９１０ＰＭ细胞系上进行实验研究。将两株细胞各随机分成６组：（１）阴性对照组（Ａ组）：只加新鲜全培养液;（２）低浓度ＣＤ３ＡＫ组（Ｂ组）：５×１０５·Ｌ－１ＣＤ３ＡＫ培养液;（３）中浓度ＣＤ３ＡＫ组（Ｃ组）：１×１０９·Ｌ－１ＣＤ３ＡＫ培养液;（４）高浓度ＣＤ３ＡＫ组（Ｃ组）：２．５×１０９·Ｌ－１ＣＤ３ＡＫ培养液;（５）顺铂阳性对照组（Ｅ组）：含１０ｍｇ·Ｌ－１顺铂培养液;（６）顺铂＋ＣＤ３ＡＫ联合用药组（Ｆ组）：含１０ｍｇ·Ｌ－１顺铂＋１×１０９·Ｌ－１ＣＤ３ＡＫ培养液,分别用ＬＤＨ活性测定、自然杀伤率及台盼蓝活细胞计数法进行了观察。结果：ＣＤ３ＡＫ细胞对卵巢癌细胞有明显的杀伤作用,两株细胞均以中浓度ＣＤ３ＡＫ细胞就显示了较好的细胞毒活性作用（Ｐ＜０．０１）,统计学上差异有高度显著性。ＣＤ３ＡＫ细胞与顺铂联合用药时,无论在细胞增     

塞来昔布抑制人胰腺癌细胞系PANC-1的增殖、侵袭、迁移能力

目的探讨COX-2抑制剂塞来昔布对人胰腺癌细胞系PANC-1增殖、侵袭、迁移能力的影响,确定塞来昔布作用的最佳浓度和最适宜的应用时间。方法不同浓度的塞来昔布(20、60和100μmol/L)处理胰腺癌细胞不同时间(24、48和72 h)后,用MTT比色法检测细胞的增殖能力;用Transwell实验检测细胞的侵袭能力;采用细胞划痕实验检测细胞的迁移能力。结果 MTT结果显示塞来昔布作用后胰腺癌细胞的增殖能力下降,呈时间和浓度依赖性(P0.05);Transwell侵袭实验结果显示塞来昔布作用后胰腺癌细胞的侵袭能力下降,呈浓度依赖性(P0.01);划痕实验结果显示塞来昔布作用后胰腺癌细胞的迁移能力下降,呈浓度依赖性(P0.01)。结论塞来昔布以浓度梯度、时间梯度的形式减弱人胰腺癌细胞系PANC-1的增殖能力,以浓度梯度的形式减弱人胰腺癌细胞系PANC-1的侵袭及迁移能力。     

表没食子儿茶素没食子酸酯与西妥昔单抗联用体内外抗食管癌细胞Eca-109的作用研究

目的研究表没食子儿茶素没食子酸酯(EGCG)与西妥昔单抗联用体内外抗食管癌Eca-109的作用。方法采用MTT法检测化合物对肿瘤细胞增殖的影响;采用克隆形成法检测EGCG对肿瘤细胞克隆形成的影响;采用流式细胞术检测化合物对肿瘤细胞凋亡的影响;采用动物实验评价药物或EGCG对移植于裸鼠的移植瘤的生长抑制作用;同时采用免疫组化的方法检测药物或EGCG对血管形成的影响。结果 EGCG能够剂量依赖性地抑制Eca-109细胞增殖,其IC50值为43.22μmol/L。经克隆形成法检测结果表明,EGCG能够明显抑制Eca-109细胞的克隆形成,IC50值为28.49μmol/L。EGCG能够显著引起Eca-109细胞凋亡,60和80μmol/L EGCG诱导的凋亡百分率分别为(21.70±0.62)%、(57.13±9.09)%。EGCG和西妥昔单抗联用对Eca-109细胞的增殖抑制作用较单独用药组强,具有相加作用。体内实验结果表明,EGCG和西妥昔单抗均能抑制Eca-109裸鼠移植瘤的生长,两者联用存在增强作用。EGCG和西妥昔单抗均能抑制裸鼠移植瘤的血管形成,两者联用血管形成抑制作用较单用组更强。结论 EGCG能够抑制食管癌Eca-109的细胞增殖和克隆形成,具有诱导Eca-109细胞凋亡作用,与西妥昔单抗联用在体内外均具有增强作用。     

The human Y chromosome suppresses the tumorigenicity of PC-3, a human prostate cancer cell line, in athymic nude mice

The loss of the Y chromosome is a frequent numerical chromosomal abnormality observed in human prostate cancer. In cancer, loss of specific genetic material frequently accompanies simultaneous inactivation of tumor suppressor genes. It is not known whether the Y chromosome harbors such genes. To address the role of genes on the Y chromosome in human prostate cancer, we transferred a tagged Y chromosome into PC-3, a human prostate cancer cell line lacking a Y chromosome. A human Y chromosome was tagged with the hisD gene and transferred to PC-3 by microcell-mediated chromosome transfer. Tumorigenicity of these PC-3 hybrids was tested in vivo and in vitro, and the results were compared with those of the polymerase chain reaction analyses conducted on the PC-3 hybrids using Y chromosome-specific markers. Among 60 mice injected with 12 different PC-3 hybrids (five mice per hybrid), tumor growth was apparent in only one mouse, whereas tumors grew in all mice injected with the parental PC-3 cells. An in vitro assay showed that the Y chromosome did not suppress anchorage-independent growth of PC-3 cells. We found that addition of the Y chromosome suppressed tumor formation by PC-3 in athymic nude mice, and that this block of tumorigenesis was independent of the in vitro growth properties of the cells. This observation suggests the presence of a gene important for prostate tumorigenesis on the Y chromosome.     

Bif-1对前列腺癌LNCap细胞生物学行为的影响

目的研究Bif-1基因的过表达对前列腺癌细胞凋亡、增殖以及迁移过程的影响。方法构建Bif-1基因全克隆并转染LNCap细胞,上调细胞中Bif-1基因的表达水平,通过流式细胞术检测细胞转染前后的细胞凋亡情况;通过MTT法检测细胞的增殖能力变化;通过划痕试验检测细胞迁移能力的变化。结果转染Bif-1基因后,Real-time PCR检测发现LNCap细胞中Bif-1基因mRNA水平△△CT高于对照组10倍以上;Western blotting条带光密度值提高2倍以上。实验组细胞凋亡比例升高至31.90%,细胞增殖能力减弱。实验组和对照组中LNCap细胞划痕愈合速度基本相同,差异无统计意义（P〉0.05）。结论 Bif-1基因在LNCap细胞中的过表达促进了细胞凋亡,抑制细胞的增殖能力,而对前列腺癌细胞的迁移能力无显著影响。基因有可能作为一个潜在的前列腺癌治疗靶点。     

RNA干扰对肝癌细胞株SMMC7721中STAT3基因表达的抑制作用

目的:研究小片段干扰RNA(siRNA)对肝癌细胞系SMMC7721的化疗敏感性.方法:根据信号转录及转录活化因子3(STAT3)基因设计siRNA序列,通过经脂质体LipofectamineTM2000以siRNA转染SMMC7721细胞.用实时定量PCR检测SMMC7721细胞中STAT3基因表达的抑制.将细胞以10 μmol/L 5-氟脲嘧啶(5-Fu)作用后,用MTT比色法检测细胞生长的抑制率.结果:成功地构建针对STAT3基因的siRNA表达载体.实时定量PCR检测结果显示,SMMC7721细胞经特异性siRNA转染后,STAT3基因的表达受到抑制.RNA干扰(RNAi)能特异、有效地抑制SMMC7721细胞中STAT3基因的表达.MTT比色法检测结果显示,经siRNA作用后SMMC7721细胞抑制率明显增加.结论:设计合成的siRNA表达载体能有效抑制STAT3基因在肝癌细胞系SMMC7721中表达,增强其对化疗药物5-FU的敏感性,为肿瘤的生物学治疗提供了实验依据.     

Cytostatic effect of phenobarbital on a human hepatoma cell line

Human hepatoma cells are considered promising for application in a bioartificial liver (BAL) support system. Since uncontrolled growth in the limited space of the bioreactor will result in cell death, regulation of growth and maintenance of liver functions are necessary for the construction of BAL systems using hepatoma cells. Substances suppressing growth and maintaining the liver functions of hepatoma cell lines were screened, and phenobarbital (PB) was found to significantly inhibit cell proliferation and DNA synthesis by the human hepatoma cell line C3A in a dose-dependent manner. Cell viability was little affected by PB treatment. Expression of the housekeeping glyceraldehyde-3-phosphate dehydrogenase and liverspecific albumin genes was not affected by PB. These results indicate that the inhibitory effect of PB on growth and protein synthesis is not related to apoptosis or necrosis. The growth-suppressing effect was maintained at least three days after the removal of PB. This kind of cytostatic substance could be used for regulation of the growth of hepatoma cell lines in the BAL system.     

HLA-DR expression in a human colonic carcinoma cell line

A human colonic carcinoma cell line (HCA-7) isolated from a well differentiated mucoid adenocarcinoma of the colon has been maintained in vitro for 3 years. It spontaneously synthesizes HLA-DR which is mainly intracytoplasmic. Stimulation with lymphocyte conditioned medium and recombinant gamma-interferon results in enhanced synthesis of HLA-DR and the appearance of the antigen on the cell surface. A dose response study showed that maximal stimulation of the culture was achieved with 50 units/ml of recombinant gamma-interferon. Staining for HLA-DR was uneven being confined to focal areas of the monolayer, which is similar to the focal expression of HLA-DR seen in sections of adenocarcinoma of the colon. The functional significance of this phenomenon is unclear, but it may explain the presence of lymphoid infiltrates in tumours.