反义肝素酶基因对胰腺癌细胞体外增殖和侵袭的抑制作用

目的:研究反义肝素酶基因对人胰腺癌SW1990细胞体外增殖和侵袭能力的抑制作用．方法:反义肝素酶基因转染胰腺癌sW1990细胞,并设空载体对照组和空白对照组,以流式细胞仪检测细胞周期;免疫组化、Western blot及RT-PCR检测肝素酶蛋白和mRNA表达;平板克隆形成实验检测细胞增殖活性,Transwell侵袭小室模型检测细胞体外侵袭能力．结果:与空白组和空载组比较,反义组细胞周期中S期比例明显减少(18.8％±2．5％vs 36．3％±2．2％,33．2％±2．1％,P<0．01),G1期细胞比例明显升高(66．0％±2．7％vs30．7％±3．2％,39．8％±4．9％,P<0．01);肝素酶蛋白及mRNA表达分别降低34．3％和37．8％:细胞克隆形成数目减少(12．2±2．8 vs 30．8±4．4,28．3±2．7,P<0．01);Transwell侵袭小室中24 h穿膜细胞数减少(13．0±3．5 vs 34．8±5．8,29．4±5．6．P<0．01)．结论:反义肝素酶基因抑制人胰腺癌SW1990细胞体外增殖及侵袭能力．     

Cox-2抑制剂NS-398对人胆管癌QBC939细胞增殖侵袭的影响

目的观察选择性Cox-2抑制剂NS-398对人胆管癌细胞株QBC939增殖、侵袭的影响。方法体外培养人胆管癌QBC939细胞,加入不同浓度的NS-398培养后,采用MTT比色法观察不同浓度NS-398作用不同时间对QBC939细胞的增殖抑制情况;采用Transwell小室法检测不同浓度NS-398作用后QBC939细胞的侵袭能力。结果 NS-398对QBC939细胞的增殖有抑制作用,并呈时间及浓度依赖性(P均<0.01),最大抑制浓度为100μmol/L。加入NS-398培养36 h后,随着药物浓度的增加,QBC939细胞体外侵袭能力明显减弱(P<0.01)。结论NS-398可抑制人胆管癌QBC939细胞的增殖,并减弱其体外侵袭能力。     

FHIT基因转染对胆管癌细胞增殖与侵袭的影响

目的:探讨FHIT基因对胆管癌细胞的增殖与侵袭的影响.方法:将含FHIT基因的重组真核表达质粒转染入胆管癌细胞株QBC939, 采用MTT实验检测转染前后细胞增殖活性, Transwell小室侵袭实验检测肿瘤细胞侵袭力.结果:转染后QBC939细胞的MTT吸光度明显下降(P <0.05), 并且转移至小室滤膜下的细胞数明显减少(48±7 vs 109±14, 104±12,均P <0.01).结论:FHIT基因能抑制胆管癌细胞株QBC939的增殖并降低其侵袭力.     

阿托伐他汀对人胆管癌QBC939细胞系侵袭的影响

目的探讨阿托伐他汀(ATV)对人胆管癌QBC939细胞系侵袭的影响及其可能作用机制。方法应用细胞培养技术培养人胆管癌QBC939细胞,经不同浓度的ATV处理后,以Matrigel侵袭实验和半定量RT-PCR检测ATV对QBC939细胞内Rho C mRNA表达的影响情况。结果 Matrigel侵袭实验显示,经10μmol/L、25μmol/L、50μmol/L干预48 h后的QBC939细胞,随着浓度的增加,其体外侵袭能力明显减弱(P0.01);半定量RT-PCR测定显示,ATV作用48 h后,QBC939细胞中Rho C的表达改变并不明显。结论 ATV可减弱人胆管癌QBC939细胞体外侵袭能力。     

野生型XPD转染对胆管癌QBC939细胞生物学行为的影响

目的:探讨野生型XPD基因对人胆管癌QBC939细胞的生物学影响. 方法:用碱裂解法提取空载质粒pEGFP-N2和重组质粒pEGFP-N2-XPD,提取出的质粒以KPNⅠ、BGIⅡ和SPHⅠ酶切鉴定.实验分4组,重组质粒pEGFP-N2-XPD组、空载质粒pEGFP-N2组、脂质体组,并用具有相同遗传背景和代数的QBC939细胞作为空白对照.用脂质体转染法瞬时转染四组细胞.荧光显微镜下观察转染后绿色荧光蛋白报告基因表达情况.提取各组细胞总RNA,合成cDNA,用聚合酶链反应(PCR)检测4组细胞中XPD、p53、cyclin D1、c-myc表达情况.并用四甲基偶氮唑盐(MTT)和流式细胞仪检测细胞增殖及其细胞周期的变化. 结果:pEGFP-N2-XPD细胞与pEGFP-N2、脂质体组和空白对照组相比,XPD mRNA表达量明显增加(0.778±0.018 vs 0.561±0.039,0.544±0.035,0.542±0.034,均P<0.01).pEGFP-N2-XPD细胞中p53 mRNA相对表达量与pEGFP-N2、脂质体组和空白对照组比较具有统计学意义(0.421±0.019 vs 0.256±0.014,0.267±0.015,0.274±0.018,均P<0.01).pEGFP-N2-XPD细胞与其他组相比,cyclin D1 mRNA相对表达量明显降低(0.339±0.041 vs 0.560±0.039,0.558±0.050,0.560±0.041,均P<0.01).pEGFP-N2-XPD细胞与其他组相比,c-myc mRNA相对表达量明显降低(0.355±0.045 vs 0.570±0.075,0.560±0.041,0.537±0.050,均P<0.01).流式细胞仪检测pEGFP-N2-XPD组细胞周期G1期为81.65%,S期为11.83%,其他组G1期分别为65.54%、56.61%、63.26%;S期分别为24.10%、29.52%、27.28%,结果具有统计学意义(P<0.05).MTT检测示pEGFP-N2-XPD细胞生长率为0.249±0.02,与其他组相比,细胞增殖力明显减弱(P<0.01). 结论:野生型XPD基因可以抑制胆管癌细胞的生长,XPD基因可抑制c-myc、cyclin D1基因的表达,增加p53基因表达.     

趋化性细胞因子受体CXCR4基因沉默对大肠癌细胞体外侵袭及增殖能力的影响

目的:研究趋化性细胞因子受体CXCR4短干扰RNA(siRNA)对大肠癌细胞系体外侵袭及增殖能力的影响.方法:利用T7 RNA聚合酶体外合成以CXCR4为靶基因的siRNA,用脂质体转染大肠癌SW480细胞,同时设立空白对照组和无关对照组.于转染后48h采用RT-PCR方法检测CXCR4 mRNA水平,免疫印迹方法检测CXCR4和MT1-MMP的蛋白质水平,Boyden小室模型检测体外侵袭能力的变化,流式细胞术检测细胞周期的分布情况,MTT法测定细胞增殖状况.结果:SW480细胞转染CXCR4 siRNA 48h后,与空白对照和无关对照相比,CXCR4 mRNA水平明显下调(51.53%±6.1% vs 78.4%±3.3%.P<0.01:51.53%±6.1% vs 87.4%±5.3%,P<0.01),CXCR4的蛋白质水平明显降低(47.3%±3.7% vs 107.2%±3.6%,P<0.01;47.3%±3.7% vs 114.7%±4.8%,P<0.01),MT1-MMP蛋白表达水平也明显下降(43.8%±2.5% vs 64.4%±4.4%,P<0.01;43.8%±2.5% vs 67.0%±2.9%,P<0.01),细胞的体外侵袭能力减弱(26.5%±6.1% vs 73.7%±3.4%,P<0.01;26.5%±6.1% vs 64.5%±5.7%,P<0.01),细胞周期的分布无明显差异.在无SDF-1存在的情况下,各组细胞的增殖无明显改变;经SDF-1刺激后,各组细胞增殖增加,但CXCR4 siRNA转染组细胞的增殖显著低于空白对照组和无关对照组(24h:0.55±0.03 vs 0.68±0.06,0.71±0.04,P<0.05;48h:0.67±0.04 vs 0.89±0.03,0.94±0.07,P<0.05;72 h:0.72±0.06 vs 1.36±0.08,1.53±0.07,P<0.01).以上各指标在空白对照和无关对照之间无显著性差异(P>0.05).结论:以CXCR4为靶向的siRNA能够有效下调CXCR4基因,降低SDF-1诱导的大肠癌细胞系体外侵袭能力及增殖海性.     

KiSS-1与肝癌细胞增殖及黏附和侵袭关系的体外实验研究

目的 观察KiSS-1基因表达对肝癌细胞体外增殖、黏附及侵袭能力的影响,为进一步探讨其抗肝细胞癌侵袭转移的机制奠定基础.方法 培养具有高转移潜能的人肝癌细胞株MHCC97-H,瞬时转染KiSS-1基因的细胞为实验组,转染空载体pcDNA3.1/HisC的细胞为空白对照组,未转染细胞为阴性对照组,采用流式细胞术与四甲基偶氮唑盐法、基质黏附实验、Transwell体外侵袭和趋化运动实验检测KiSS-1表达对MHCC97-H细胞体外增殖、黏附、侵袭和运动能力的影响.应用SPSS13.0统计软件包进行统计分析,组间差异以两样本t检验分析. 结果 转染组、空载体组和未转染组与Matrigel基质的黏附能力(A值)分别为0.257±0.029、0.374±0.016和0.394±0.031,转染组明显低于空载体组(t=-7.90345,P＜0.01)和未转染组(t=-7.22752,P＜0.01);与Fibronectin基质的黏附能力(A值)分别为0.292±0.004、0.394±0.010和0.412±0.023,转染组明显低于空载体组(t=-20.93138,P＜0.01)和未转染组(t=-11.31371,P＜0.01);趋化运动至Transwell小室下室表面的细胞数分别为65.80±1.92、93.80±2.28和96.40±2.07,转染组明显低于空载体组(t=-30.11750,P＜ 0.01)和未转染组(t=-24.19142,P＜0.01);侵袭至Transwell小室下室表面的细胞数为42.40±1.14、66.00±1.58和67.80±1.92,转染组明显低于空载体组(t=-27.0711,P＜0.01)和未转染组(t=-25.4,P＜0.01).而转染组、空载体组和未转染组的细胞增殖能力(A值)分别为0.644±0.027、0.669±0.022和0.678±0.027,转染组略低于空载体组(t=-1.60371,P＞0.05)和未转染组(t=-1.97828,P＞0.05);同时,转染组较空载体组和未转染组未出现明显的G1期阻滞和S期阻滞,也未出现明显的凋亡峰.结论 KiSS-1表达虽不影响肝癌细胞的体外增殖能力,但可抑制其黏附、侵袭和运动能力,提示KiSS-1可作为一个候选的肝细胞癌转移抑制基因,可望成为肝癌侵袭转移治疗的新靶点.     

RNA干扰阻断cyclin D1表达抑制结肠癌细胞增殖

目的:使用RNA干扰技术阻断人结肠癌细胞cyclin D1的表达,检测其对细胞生长增殖的影响和机制.方法:将人结肠癌细胞株HT-29分成3组,antisense组(转染反义链组)、sense组(转染正义链组)和对照组.免疫沉淀和蛋白质印迹法观察cyclin D1 siRNA对细胞cyclin D1蛋白质表达的影响,并对蛋白质电泳图像进行分析,MTT比色法绘制细胞生长曲线,~3H-TdR技术和流式细胞技术观测细胞周期变化结果:反义siRNA有效抑制了cyclin D1蛋白质表达,MTT实验显示,转染反义siRNA的细胞,生长代谢减慢,与对照组细胞差异显著(P<0.01).而antisense组细胞24 h的~3H-TdR渗入量,G_0/G_1期和S期细胞较对照组和sense组明显减少(~3H-TdR:1181.8±117.97 vs 1798.4±55.36,1851.4±83.46:P<0.01;G_0/G_1期:79.31% vs 60.87%.59.14%:S期:13.67% vs 26.42%,27.93%:P<0.01).结论:RNA干扰技术能有效减弱cyclin D1蛋白质表达,使细胞周期受阻,并抑制结肠癌细胞的生长增殖.     

HBx基因下调p21对HepG2细胞增殖与凋亡的影响

目的:构建转基因细胞模型HepG2/HBx,观察HBx基因对HepG2细胞增殖、周期和凋亡的影响, 探讨细胞周期蛋白P21在其中的作用和意义.方法:应用脂质体转染和G418筛选构建稳定表达HBx的转基因细胞HepG2/HBx,RT-PCR和Western blot鉴定HBx mRNA与蛋白的表达.分别以四唑蓝(MTT)比色法、流式细胞术检测HepG2/HBx细胞及对照组HepG2与HepG2/pcDNA3.1细胞(转染空载体pcDNA3.1的HepG2细胞)的增殖、周期和凋亡.另半定量RT-PCR检测各组细胞中细胞周期蛋白P21与抑癌基因p53mRNA的表达.结果:HepG2/HBx细胞中有HBx mRNA和蛋白的表达.HepG2/HBx细胞生长速度加快.HepG2/HBx中G0/G1期细胞比例较对照组显著减少(43.34%±3.11%vs57.69±4.28%,P<0.01),S期细胞比例明显增加(28.69%±1.17%vs22.41%±1.99%,P<0.05),同时还发现与对照组相比其凋亡率也显著降低(1.19%±0.06%vs 5.43%±0.42%, P<0.001).细胞周期蛋白p21 mRNA在HepG2/HBx细胞中的表达较对照组细胞显著降低(0.16±0.05vs0.78±0.15,P<0.001),而p53表达则无显著变化.结论:HBx基因可下调细胞周期蛋白P21mRNA的表达,可能参与HBx基因加速HepG2细胞周期进程、促进细胞增殖以及抑制细胞凋亡的作用.     

WWOX基因转染对胆管癌细胞增殖、凋亡及侵袭的影响

目的:探讨WWOX基因转染胆管癌细胞株QBC939后对其增殖、凋亡与侵袭性的影响.方法:用脂质体转染法将WWOX重组真核表达质粒转染QBC939细胞,建立稳定表达WWOX基因的细胞株.将其分为以下3组:QBC939组,QBC939/con组和QBC939/WWOX组.荧光定量RT-PCR和Western blot法检测...     

Degradation of human calcitonin in human plasma

The degradation in vitro of human calcitonin (HCT) in plasma from normal subjects and patients with disorders of calcium metabolism was studied. As measured by radioimmunoassay, the hormone was stable at 37°C in phosphate buffer for 24 hr but progressively disappeared when incubated in normal human plasma. The loss was temperature and pH dependent, being maximal at 37°C between pH 6.0 and 7.0. Under these conditions ¹²⁵I-HCT appeared to be degraded to at least one labeled fragment that behaved on polyacrylamide gels as if it were smaller than 1000 daltons. The rate of formation of this fragment correlated well with the rate of loss of immunoreactive HCT. Compared to plasma from normal individuals, plasma from patients with hypercalcemia due to causes other than hyperparathyroidism degraded HCT significantly more rapidly. The mean loss of HCT in 5 hr in plasma from nine such patients was 54% ± 17% (± 2 SEM), compared to 22% ± 5% in plasma from 22 healthy volunteers (p < 0.001). Those patients whose plasma most rapidly degraded HCT had milkalkali syndrome, metastatic carcinoma of the colon, metastatic oat cell carcinoma of the lung, and metastatic breast carcinoma. Rates of HCT degradation in plasma from eight hypercalcemic patients with hyperparathyroidism and seventeen normocalcemic patients with malignancy were within the normal range. From these findings we conclude that human plasma contains one or more enzymes that degrade HCT and that the hormone is degraded more rapidly in plasma from some patients with hypercalcemia.     

Interaction of human chorionic gonadotropin and human luteinizing hormone with human thyroid membranes

In an attempt to identify a possible pathogenetic role for the hCG molecule in the mechanism of the hyperthyroidism which occurs in choriocarcinoma, we have looked for evidence that the hCG molecule has a thyrotropic action on the human thyroid. The thyrotropic activity of various hCG preparations on the human thyroid was assessed by measuring the stimulation of adenylate cyclase activity in human thyroid plasma membranes purified by sucrose density gradient centrifugation. The highly purified hCG CR119 preparation stimulated human thyroid adenylate cyclase activity. Its activity was more than 654 times greater than could be accounted for by human TSH (hTSH) contamination of the preparation, as determined by RIA. The thyrotropic activity intrinsic to 1.0 IU hCG was equivalent to roughly 0.27 microU hTSH. Significant saturable binding of the 125I-labeled highly purified hCG preparation to human thyroid membranes was demonstrated, and the bound component was characterized. Its apparent molecular size, subunit composition, and testis receptor-binding characteristics were those of the hCG molecule. Examination of a crude urinary hCG preparation in adenylate cyclase and TSH radioligand assays using human thyroid membranes showed no evidence of any molecule other than hCG with a thyrotropic action on the human thyroid. Given that hCG binds to and stimulates adenylate cyclase activity in human thyroid tissue, as the above data indicate, then human LH (hLH) would be expected to do the same, since hLH and hCG have such strong structural and functional similarities. As anticipated, a highly purified hLH preparation exhibited TSH binding inhibition and adenylate cyclase stimulation. Its activity was more than 1030 times greater than could be accounted for by hTSH contamination of the preparation. The thyrotropic activity intrinsic to 1.0 IU hLH was equivalent to roughly 44 microU hTSH. Thus, in addition to other shared properties, the hLH molecule and the hCG molecule share the ability to interact with human thyroid tissue. These results strongly indicate that the hCG molecule has a thyrotropic action on the human thyroid and support the hypothesis that hCG is the thyrotropic factor that mediates the hyperthyroidism which occurs in patients with hCG-secreting neoplasms.     

Degradation of human calcitonin in human plasmas.

The degradation in vitro of human calcitonin (HCT) in plasma from normal subjects and patients with disorders of calcium metabolism was studied. As measured by radioimmunoassay, the hormone was stable at 37°C in phosphate buffer for 24 hr but progressively disappeared when incubated in normal human plasma. The loss was temperature and pH dependent, being maximal at 37°C between pH 6.0 and 7.0. Under these conditions ¹²⁵I-HCT appeared to be degraded to at least one labeled fragment that behaved on polyacrylamide gels as if it were smaller than 1000 daltons. The rate of formation of this fragment correlated well with the rate of loss of immunoreactive HCT. Compared to plasma from normal individuals, plasma from patients with hypercalcemia due to causes other than hyperparathyroidism degraded HCT significantly more rapidly. The mean loss of HCT in 5 hr in plasma from nine such patients was 54% ± 17% (± 2 SEM), compared to 22% ± 5% in plasma from 22 healthy volunteers (p < 0.001). Those patients whose plasma most rapidly degraded HCT had milkalkali syndrome, metastatic carcinoma of the colon, metastatic oat cell carcinoma of the lung, and metastatic breast carcinoma. Rates of HCT degradation in plasma from eight hypercalcemic patients with hyperparathyroidism and seventeen normocalcemic patients with malignancy were within the normal range. From these findings we conclude that human plasma contains one or more enzymes that degrade HCT and that the hormone is degraded more rapidly in plasma from some patients with hypercalcemia.     

Effect of human recombinant Endostatin protein on human angiogenesis

Tumor growth and metastasis are dependent on the development of new blood vessels. Inhibitors of new vessel growth have been widely investigated as anti-tumor agents. Endostatin, a 20 kDa C-terminal fragment of collagen XVIII inhibits endothelial cell proliferation, induces endothelial cell apoptosis, and can both inhibit and reverse tumor growth in mice. However, human recombinant endostatin has had limited testing against human tissue targets. To investigate the effect of human endostatin on a human vessel target over a broad range of concentrations (10^–12–10^–4 M), human placental vein disks were grown for a period of 2 weeks in a 0.3% fibrin clot overlayed with growth medium. Disks from five individual placentas were tested. For each placenta utilized, a control (medium and 20% fetal bovine serum [FBS]) group and a group treated with heparin (300 g/ml) and hydrocortisone 21-phosphate (350 g/ml) (heparin-steroid) at a dose known to inhibit angiogenesis were included. Endostatin was tested at concentrations of 10^–12–10^–4 M in medium containing 20% FBS. The rate of initiation and the angiogenic growth index (on a visually graded semi-quantitative scale of 0–16) were determined for all experimental conditions. Endostatin inhibited angiogenesis in our model only in high concentrations. At 10^–5 M, endostatin did not alter the percent of wells that initiated an angiogenic response, but significantly inhibited subsequent vessel growth. At 10^–4 M, endostatin was able to inhibit both initiation and subsequent new vessel growth. Human endostatin can inhibit the initiation of a human angiogenic response and inhibit the subsequent proliferation of human neovessels when used at high doses in a continuous exposure model.     

Absence of a directly causative role for human herpesvirus 7 in human lymphoma and a review of human herpesvirus 6 in human malignancy

 In search of a (new) viral etiological agent, we screened 64 lymph node samples from Hodgkin's disease (HD) and 43 samples (32 lymph node and 11 skin biopsies) from non-Hodgkin's lymphoma (NHL) for human herpesvirus 7 (HHV-7). Twenty-nine control samples were tested as well, including 17 with benign lymphadenopathy. None of the samples tested positive by Southern blot hybridization using HHV-7-specific probes. We conclude that there is no major HHV-7 load in human lymphoma and that HHV-7 is not likely to be directly involved in its etiology. This is in contrast to a small minority of human lymphoproliferative diseases in which HHV-6 can be found at high copy number, but in which an etiological role is still uncertain. Received: September 8, 1998 / Accepted: November 2, 1998     

Comparison of recombinant human thrombin and plasma-derived human alpha-thrombin

Bleeding can be a serious complication of surgery, and topical thrombin is widely used as an adjunct to hemostasis in diverse surgical settings. The potent hemostatic properties of thrombin derive from its ability to activate platelets directly to aggregate and adhere to damaged vessels and to catalyze the formation simultaneously of a fibrin matrix. Application of exogenous thrombin bypasses the physiological process of generating a thrombin burst by directly initiating the terminal reactions of blood clot formation. Currently, thrombin used to control surgical bleeding is primarily from bovine plasma, with a small percentage from human plasma. Human thrombin isolated from pooled plasma carries the risk of transmitting plasma-borne pathogens or prion diseases. The bovine preparations have been associated with protein and preparative contaminants that pose potential risks of developing cross-reacting antibodies. There is a need for a pure therapeutic preparation of human thrombin. Recombinant human thrombin (rhThrombin) has been efficiently produced from a prethrombin-1 precursor obtained from Chinese hamster ovary cell culture. This rhThrombin is substantially free of process-derived contaminants and has been characterized extensively in terms of composition, primary, secondary, and tertiary structure, enzymatic activity; and in vivo pharmacology. In vivo studies of topically applied rhThrombin have shown it is effective in achieving hemostasis in a rabbit liver excisional wound model. Clinical studies are ongoing to evaluate the safety and efficacy of rhThrombin as an adjunct to hemostasis in patients undergoing surgery.     

Effects of zymosan-activated human granulocytes on isolated human airways

In asthma a temporal association exists between the late allergic reaction (LAR), the influx of granulocytes into the airway wall, and an increase in bronchial responsiveness. We therefore tested the hypothesis that activated human granulocytes constrict isolated human airways and increase their sensitivity to cholinergic stimuli. Bronchial rings were dissected from 23 lung tissue specimens collected at thoracotomy and studied isotonically in organ baths. Airways were incubated with 1, 2, 5, 10, or 20 x 10(6) granulocytes from normal or atopic donors. Activation of the cells with serum-treated zymosan (STZ, 0.2 mg/ml), which itself did not alter baseline airway caliber, resulted in a bronchoconstriction proportional to the number of zymosan-activated granulocytes (ZAG) present (rs = 0.79, p less than 0.001). This contraction was reduced by about 70% with the leukotriene C4/D4 receptor antagonist FPL 55712 (11.5 microM; p less than 0.001) or with the lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM; p less than 0.001). The scavengers of activated oxygen molecules superoxide dismutase (300 U/ml) and bovine catalase (5,000 U/ml), the cyclooxygenase inhibitor indomethacin (10 microM), or the histamine (H1) receptor antagonist mepyramine (2.8 microM) had no effect. Granulocyte suspensions from atopic donors contained more eosinophils (p less than 0.001), and the magnitude of the contraction to 10 x 10(6) ZAG was related to the proportion of eosinophils (rs = 0.66, p less than 0.01). The sensitivity of the airways to methacholine was unchanged in the presence of 1, 2, or 5 x 10(6) ZAG and decreased with 10 or 20 x 10(6) ZAG (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)