125I-脱氧尿嘧啶核苷对重组质粒pcDNAEgr-IFNγ体外辐射诱导表达的作用

目的研究125I-脱氧尿嘧啶核苷对重组质粒pcDNAEgr-IFNγ在体外培养B16细胞中的辐射诱导表达作用。方法以脂质体介导法将pcDNAEgr-IFNγ重组质粒转染B16细胞,48h后,于培养液中加入125I-UdR,使其终放射性浓度分别为0(空白对照)、1、5、10、20、50kBq/ml,24h后,采用ELISA法定量检测细胞上清中IFNγ。结果转染组细胞IFNγ蛋白表达随着细胞培养液中125I-UdR浓度的增高而增高,放射性浓度为5、10、20、50kBq/ml组IFNγ的蛋白表达明显高于0kBq/ml组(P<0.05~0.001),其中50kBq/ml组表达量最高,为0kBq/ml组的3.66倍;加入50kBq/ml125I-UdR后6h上清中IFNγ表达量即明显升高(P<0.05~0.001),并随时间延长逐渐增加,照后48h达峰值,表达量为未加入125I-UdR时的6.51倍。结论pcDNAEgr-IFNγ重组质粒在125I-UdR诱导下可有效表达IFNγ,并且其辐射诱导IFNγ基因表达增强特性具有一定的量效和时程规律。     

问号钩端螺旋体毒力基因mviN的表达及细胞毒性作用研究

目的克隆表达问号钩端螺旋体毒力基因mviN并观察表达产物对血管内皮细胞ECV304及肺上皮细胞A549的毒性作用。方法将mviN基因插入原核表达载体pET32a(+),构建重组质粒pET-mviN,转化大肠杆菌BL21(DH3)以高效表达携带组氨酸标签的Trx-MviN融合蛋白,并作亲和层析纯化。以XTT法检测毒力蛋白Trx-MviN对ECV304及A549细胞增殖的抑制作用,同时以流式细胞术检测其作用于ECV304及A549后的细胞凋亡率。结果成功构建了重组质粒pET-mviN并高效表达出Trx-MviN融合蛋白;毒力蛋白Trx-MviN作用于ECV304及A549后,与对照组相比较,其细胞增殖被显著抑制(P<0.05),而细胞凋亡率显著增加(P<0.01)。结论重组质粒pET-mviN高效表达出Trx-MviN融合蛋白,纯化后的融合蛋白Trx-MviN对ECV304及A549具有细胞毒性作用。     

3-BrPA对肺癌A549/DDP细胞耐药的逆转作用研究

目的探究3-Br PA对肺癌A549/DDP细胞耐药的逆转作用及其机制。方法 1细胞培养:RPMI1640培养基培养肺癌A549细胞及人肺腺癌耐顺铂细胞株A549/DDP细胞;2采用CCK8及Western blot法观察3-Br PA对A549/DDP细胞的耐药逆转作用和P-糖蛋白(P-glycoprote,P-gp)的表达水平;采用流式细胞术检测不同浓度3-Br PA作用A549/DDP细胞后细胞内罗丹明-123(Rhodamine-123,Rh123)的蓄积情况。结果顺铂对A549细胞、A549/DDP细胞及3-Br PA作用后的A549/DDP细胞的IC50分别为6.714μg/ml、38.99μg/ml及12.86μg/ml,逆转倍数为3.03,相对逆转效率为81%。3-Br PA可使A549/DDP细胞的P-gp表达下调。3-Br PA可使A549/DDP细胞内的Rh123蓄积增多并呈剂量依赖性。结论 3-Br PA可逆转A549/DDP细胞对顺铂耐药,作用机制可能与其抑制P-gp的功能和表达有关。     

大蒜素对人肺腺癌细胞生长抑制作用的实验研究

目的研究大蒜素对人肺腺癌A549细胞增殖抑制、细胞凋亡的影响。方法用不同浓度大蒜素作用体外培养的A549细胞,采用CCK8法检测大蒜素对A549细胞增殖抑制能力,荧光显微镜观察A549细胞形态学变化,ELISA法测定A549细胞上清液VEGF水平,比色法检测caspase3、caspase9活性变化。结果大蒜素对A549细胞增殖有明显抑制作用,并呈时间及浓度依赖性;24 h、48 h及72 h大蒜素对A549细胞的IC50分别为114.26μg/ml、85.27μg/ml、76.83μg/ml(P<0.05);60μg/ml以上大蒜素作用A549细胞48h可产生显著凋亡特征;随大蒜素浓度增加,A549细胞上清液VEGF水平呈下降趋势,A549细胞caspase3、caspase9蛋白活性显著增加。结论大蒜素能显著抑制A549细胞增殖、诱导其凋亡,可能与VEGF表达下降及caspase3、caspase9激活相关。     

反应停对肺腺癌细胞株诱导的血管内皮细胞增殖的影响

目的探讨反应停对肺腺癌细胞株诱导的血管内皮细胞增殖的影响。方法用噻唑兰法检测不同浓度反应停对血管内皮细胞株ECV304（ECV304细胞）、肺腺癌细胞株SPC—A1（SPC—A1细胞）及其诱导的血管内皮细胞ECV304增殖的影响。结果反应停浓度为8．0和80．0μg／ml时对ECV304细胞增殖有抑制作用（P〈0．05）;不同浓度反应停对SIC-A1细胞增殖无影响;经8．0和80．0μg／ml反应停作用后SIC-A1细胞条件培养液对ECV304细胞增殖有抑制作用（P均〈0．05）,其细胞生长抑制率分别为5．19％、12．49％。结论反应停对SPC-A1细胞诱导的ECV304细胞增殖有抑制作用。     

糖基化终产物对人血管内皮细胞基质金属蛋白酶-2表达和活性的影响

目的探讨糖基化终产物(AGEs)对人血管内皮细胞ECV304基质金属蛋白酶-2(MMP-2)表达和活性的影响。方法应用AGEs作用ECV304细胞后,采用RT-PCR法及明胶酶图分析方法分析MMP-2表达和活性与AGEs浓度和时间的关系。结果 RT-PCR结果显示,AGEs能够诱导ECV304细胞表达MMP-2,其诱导表达具有浓度和时间依赖性(P<0.01)。明胶酶图分析显示,MMP-2降解明胶的活性与AGEs具有浓度依赖性。结论 AGEs能够促进ECV304细胞MMP-2的表达和活性,推测AGEs调控MMP-2表达和活性可能在血管壁内皮细胞影响动脉粥样硬化的进展中发挥一定的作用。     

白藜芦醇诱导肺腺癌A549细胞凋亡的机制

目的探讨白藜芦醇(Res)诱导肺腺癌A549细胞凋亡的机制。方法用不同浓度Res处理A549细胞24 h后,倒置显微镜观察细胞形态学变化,4,6-二氨基-2-苯基吲哚(DAPI)细胞核染色后荧光显微镜观察细胞凋亡特征,噻唑蓝(MTT)检测对于A549细胞的抑制生长作用,Western印迹法检测Res作用A549细胞后P53、Bax、Bcl-2、Cleaved-Caspase3蛋白的表达变化。结果 Res处理A549细胞后,细胞间隙变大,细胞核分裂,随着药物浓度的增加上述形态变化明显。25²00μmol/L浓度的Res可明显抑制A549细胞的增殖,具有剂量依赖性,24 h的IC50为100μmol/L,而且细胞内的P53、Bax、Cleaved-Caspase3蛋白表达量升高,Bcl-2蛋白被抑制,Bcl-2/Bax比值下降。结论 Res抑制A549细胞增殖,并通过P53途径诱导A549细胞凋亡,Bax、Bcl-2和Cleaved-Caspase3参与了凋亡过程。     

达肝素钠对A549细胞生长状态及细胞周期的影响

目的研究达肝素钠对体外生长的肺腺癌A549细胞活性及细胞周期的影响。方法用6种不同浓度(0、2、10、50、100和500IU/ml)的达肝素钠干预A549细胞的生长,用MTT方法在4个时间点(6、12、24和36h)检测细胞活性;将经过24h普通培养的A549细胞继续在含有50IU/ml达肝素钠的培养液中培育,以正常培育的细胞作为对照组,将两组细胞24h后以PI染液染色,用流式细胞仪检测并分析细胞周期。结果达肝素钠降低A549细胞活性,这种作用表现出时间依赖性和浓度依赖性;达肝素钠主要抑制细胞在G1期。结论达肝素钠对A549细胞活性的降低与对细胞周期的抑制作用有关。     

不同浓度肿瘤坏死因子-α对肺腺癌A549细胞的影响

目的 观察不同浓度肿瘤坏死因子-α(TNF-α)对肺腺癌A549细胞产生的保护、促炎、促细胞凋亡效应.方法 分别以0、1、10、25、50 ng/mL的TNF-α诱导肺腺癌A549细胞6h.采用MTT比色法观察细胞存活率;应用活性氧(ROS)荧光探针DCFH-DA检测ROS强度;RT-PCR法检测Nrf-2、NF-κB基因的表达;DAPI细胞核荧光染色法处理细胞,荧光显微镜下观察细胞凋亡.结果 0、1、10、25、50 ng/mL TNF-α对肺腺癌A549细胞存活率无影响(P均＞0.05);100 ng/mL TNF-α诱导后,与对照组比较,细胞存活率降低(P＜0.05).随着TNF-α浓度增加,肺腺癌A549细胞产生ROS的量随之增加(P均＜0.05),Nrf-2 mRNA表达减少(P均＜0.05),NF-κB mRNA表达增加(P均＜0.05),细胞凋亡率有上升趋势.结论 不同浓度TNF-α诱导肺腺癌A549细胞可产生保护、促炎、促细胞凋亡等不同细胞效应,且随TNF-α浓度增加,细胞损伤加重;其机制可能与ROS产生的量有关.     

索拉非尼对人肺腺癌细胞株A549抑制作用的研究

目的探讨索拉非尼（Sorafenib）在体外对人肺腺痛细胞株A549增殖、凋亡的影响。方法采用MTT法检测Sor-afenib对A549细胞增殖抑制率,应用流式细胞仪检测细胞凋亡率。结果Sorafenib在1．5～12．20μmol／L浓度范围内能明显抑制A549细胞的增殖,此抑制作用呈时间-剂量依赖效应（P〈0．05）,而不同药物浓度的Sorafenib作用于A549细胞72h,显著增加其凋亡率,呈剂量依赖性,差异有显著性（P〈0．05）。结论Sorafenib能抑制人肺腺癌细胞株A549细胞增殖,呈时间-剂量依赖效应,还能促进A549细胞凋亡,亦呈剂量依赖性。     

载体肝干细胞分泌表达的Endostatin对血管内皮细胞增殖和凋亡的影响

目的观察携带内皮抑素基因的载体肝干细胞分泌表达的内皮抑素蛋白（Endostatin，ES）对血管内皮细胞体外增殖和凋亡的影响。方法体外扩增培养本室构建的携带内皮抑素基因的载体肝干细胞WB-ES，收集合有分泌型Endostatin的上清液。将人脐静脉内皮细胞ECV304体外增殖培养，加入不同浓度的含倍比稀释Endostatin的上清液，在不同的作用时间（24、48、72h），通过四甲基偶氮唑蓝比色法（MTT）检测细胞生长抑制率。采用流式细胞仪检测细胞周期和凋亡率，分析载体肝干细胞WB—ES分泌表达的Endostatin对ECV304的增殖和凋亡的影响。结果载体肝干细胞胞外表达的Endostatin对人脐静脉内皮细胞ECV304生长有显著抑制作用，48h作用达到高峰，随浓度增加抑制作用增强；Endostatin作用的实验组，G1期细胞比例增加，s期细胞数下降，抑制细胞生长的机制可能主要是通过对细胞增殖的影响（P〈0．05）；实验组细胞存在细胞凋亡增加现象，但与对照组比较凋亡率差异无统计学意义。结论携带内皮抑素基因的载体肝干细胞WB-ES胞外表达的Endostatin在体外可有效抑制血管内皮细胞增殖，载体肝干细胞WB-ES有望成为靶向抗肿瘤血管生成的肝癌基因治疗载体细胞。     

不同浓度葡萄糖对人血管内皮细胞的影响

目的研究高浓度葡萄糖对人血管内皮细胞的影响。方法在人脐静脉内皮细胞株ECV304培养基中分别加入5.5、20、40 mmol/L葡萄糖,作用24~72 h。采用四甲基偶氮唑盐微量酶反应比色（MTT）法观察细胞增殖情况,倒置相差显微镜观察细胞形态,透射电镜观察内皮细胞V304凋亡情况;流式细胞术检测细胞凋亡率。结果高浓度葡萄糖能明显抑制血管内皮细胞的增殖,诱导培养的内皮细胞发生凋亡,细胞凋亡率增加,并随浓度的增高、时间的延长作用明显。结论高浓度葡萄糖能抑制培养的人血管内皮细胞增殖,促进细胞凋亡。     

Non-Specific and Specific Effects of a Granulocytic Bio-Inhibitor on Different Haematopoietic Cell Populations

The effect of a leucocyte conditioned medium derivative on the 5-¹²⁵iodo-2′-deoxy-uridine (¹²⁵I-UdR) uptake by 6 haematopoietic cell populations with a simple in vitro assay system was tested. The low doses of the test substance - a putative granulocytic chalone - inhibited significantly the uptake of ¹²⁵I-UdR only in predominantly granulocytic cell cultures. This inhibition was achieved using μg/ml concentrations, which have previously been shown to be inhibitory to normal and malignant granulocytic cells in vivo. The test preparations inhibited the uptake of ¹²⁵I-UdR in all cell populations (non-granulocytic included) when doses exceeding 10 μg/ml were used. The prerequisite for an adequate demonstration of the specificity of a bio-inhibitor is discussed.     

血管内皮生长因子反义寡核苷酸抑制甲状腺癌血管生成的效应

目的探讨反义寡核苷酸(ASODN)抑制甲状腺癌细胞血管内皮生长因子(VEGF)表达及内皮细胞生长的效应。方法设计合成靶向VEGF的ASODN转染人髓状甲状腺癌细胞系(TT)细胞,并制备相应条件培养基作用内皮细胞ECV304,设正义寡核苷酸(SODN)和空白对照组进行比较。观察细胞生长状态,RT PCR、免疫细胞化学法检测TT细胞VEGFmRNA和蛋白表达,四氮唑蓝法检测TT和ECV304细胞生长抑制率(IR),流式细胞仪、吖啶橙/溴化乙锭染色法检测ECV304细胞凋亡状态。结果ASODN组TT细胞VEGFmRNA和蛋白表达显著低于SODN和对照组(P<0.01),但IR差异无统计学意义(P>0.05);各转染组ECV304细胞IR差异亦无统计学意义(P>0.05);而经各ASODN组TT细胞条件培养基作用的ECV304细胞生长明显受抑,IR(分别为0.21±0.03、0.31±0.01、0.42±0.22)显著高于SODN组(0.05±0.03,P<0.01),并伴明显细胞凋亡,上述效应呈浓度依赖性。结论ASODN可通过特异性封闭甲状腺癌细胞VEGF表达,抑制内皮细胞生长,干扰肿瘤血管生成。     

Hormonal regulation of the NF-kappaB signaling pathway

Studies with mammalian vascular cells have suggested growth inhibitory effects of estrogen on the vascular wall. To investigate the involvement of estrogen receptor-alpha (ER) in the control of endothelial cell proliferation, we have stably transfected human estrogen receptor-alpha cDNA into the endothelial cell line ECV304. The clone ECV-ER, thus obtained, over-expresses estrogen receptor to a level approximately 10-fold higher than the parent cell line. Effects of this over-expression were studied on the cell growth rate, and on the levels of secreted endothelin-1 and vascular endothelial growth factor (VEGF). Similar to the previously reported data in other cell types, we found the transfection of ER in ECV304 cells to be inhibitory to their growth. Our ER-over-expressing clone of ECV304 also showed an inhibition of secreted endothelin-1 and VEGF levels. Moreover, the growth inhibition of this ER-over-expressing clone was reversed by the addition of endothelin-1 or VEGF to the medium. In view of the growth-stimulatory effect of endothelin-1 and VEGF on vascular cells, our results indicate that estrogen receptor-alpha may bring about its growth inhibition partly by suppressing endothelin-1 and/or VEGF production in ECV304 cells.     

TGFbeta1 is involved in high glucose-induced accumulation of pericellular chondroitin sulphate in human endothelial cells

High glucose-induced endothelial cell dysfunction is considered to be the main cause of the development of vascular diabetes complications. Cultured endothelial cells exposed to high glucose in vitro demonstrate a variety of alterations, including extracellular matrix (ECM) deposition, growth inhibition, and changes in cell motility. Some of these effects were shown to be mediated by the up-regulation of endothelial transforming growth factor-beta1 (TGFbeta1) secretion and activation. We investigated the influence of high glucose on human immortalized endothelial cell line ECV304. According to our data, confluent cells exposed to 30 mM glucose for 48 h secrete the increased amount of total and active TGFbeta1 ( approximately 1.4-fold), and accumulate more chondroitin sulphate (CS) in their conditioned medium, pericellular matrix, and cell layer ( approximately 1.6- to 2.0-fold). By blocking the coupling of CS chains to the core protein with p-nitrophenyl-beta-D-xyloside and by chondroitinase ABC treatment, we demonstrated that the increased accumulation of pericellular CS is accompanied by increased cell attachment to immobilized hyaluronic acid (HA), while the expression of cell surface CD44 remains unaltered. Since the exogenous TGFbeta1 affects ECV304 cells in a similar manner, and anti-TGFbeta1-neutralizing antibody cancels the effect of high glucose, we suggest the involvement of TGFbeta1 in the development of endothelial cell response to high glucose in terms of CS accumulation and cell binding to HA.     

Non-specific and specific effects of a granulocytic bio-inhibitor on different haematopoietic cell populations.

The effect of a leucocyte conditioned medium derivative on the 5-125iodo-2'-deoxyuridine (125I-UdR) uptake by 6 haematopoietic cell populations with a simple in vitro assay system was tested. The low doses of the test substance--a putative granulocytic chalone--inhibited significantly the uptake of 125I-UdR only in predominantly granulocytic cell cultures. This inhibition was achieved using micrograms/ml concentrations, which have previously been shown to be inhibitory to normal and malignant granulocytic cells in vivo. The test preparations inhibited the uptake of 125I-UdR in all cell populations (nongranulocytic included) when doses exceeding 10 micrograms/ml were used. The prerequisite for an adequate demonstration of the specificity of a bio-inhibitor is discussed.     

Transcellular iodide transport and iodination on the apical plasma membrane by monolayer porcine thyroid cells cultured on collagen-coated filters

Isolated porcine thyroid follicular cells were cultured on a collagen-coated Millipore filter to form a monolayer. The monolayer could translocate 125I added in the medium beneath the filter (basal medium) into the medium above the monolayer (apical medium) and form an iodide concentration gradient of several-fold. Transcellular iodide pump activity was observed when the cells were cultured with TSH in the basal medium. In the absence of TSH, the translocation of iodide was very slow. The concentration of TSH required to activate the iodide pump was 0.1-0.3 mU/ml. Addition of ClO4- to the basal medium inhibited transcellular transport, whilst addition of ClO4- to the apical medium was much less effective. Constituents labelled with 125I in the apical medium were analysed. The amount of protein-bound 125I measured by acid precipitation was 3-8% of the total radioactivity. The residual radioactivity was found to be iodide ion by paper chromatography. Further analysis by sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed that most of the 125I-labelled protein was at the position of bovine serum albumin which had been added to the culture medium. The monolayer culture of cells on collagen-coated filter would be a useful experimental system for analysing thyroid cell functions for which the cell polarity is essential.     

土贝母苷甲对人肺腺癌细胞增殖及凋亡的影响

目的 探讨土贝母苷甲对人肺腺癌细胞(A549)增殖及凋亡的影响.方法 应用MTS法检测土贝母苷甲不同浓度、不同时间对A549细胞增殖的影响;使用1μg/mL土贝母苷甲处理A549细胞48 h作为处理组,对照组加入培养基培养48h;使用流式细胞仪分别检测两组细胞的周期分布及细胞凋亡率;并使用Western blot检测两...