人骨肉瘤细胞株143B转移和浸润中双氢青蒿素抑制作用的研究

目的：研究双氢青蒿素（Dihydroarteminin,DHA）对人骨肉瘤细胞株143B转移和浸润的影响并对其机制进行探讨。方法：通过划痕愈合试验、Traaasweu穿梭试验、人工基质胶浸润试验,检测DHA对143B细胞的转移迁徙能力的影响;WesternB｜oting检测与人骨肉瘤细胞株143B转移和浸润密切相关的两种蛋白：基质金属蛋白酶（MatrixMetalProteinase9,MMP-9）和血管内皮生长因子（VascularEndothelialGrowthFactor,VEGF）,经DHA作用后表达的影响。结果：划痕愈合试验、Transwdl穿梭试验和基质胶侵袭试验结果均呈现阳性,表明DHA可有效地抑制人骨肉瘤细胞143B的转移与浸润;westerlaBloting结果显示MMP-9和VEGF的表达受到明显抑制,抑制效果随着其浓度增大和时间延长而增加。结论：双氯青蒿素具有明显的抑制人骨肉瘤细胞143B的转移和浸润的作用,可能与双氢青蒿素能抑制MMP-9和VEGF表达有关。     

双氢青蒿素诱导前列腺癌PC-3细胞凋亡的形态学研究

目的：研究双氢青蒿素（DHA）诱导前列腺癌PC-3细胞凋亡作用,并观察其形态学变化。方法：以前列腺癌PC-3细胞为研究对象,在体外经不同浓度的DHA处理48小时后,扫描电镜（SEM）观察细胞凋亡;并建立PC-3细胞裸鼠种植瘤模型,20只模型鼠随机分为对照组、溶剂组、DHA大剂量组及DHA小剂量组,经13天干预后,透射电镜（TEM）观察肿瘤组织形态学变化,体视学分析肿瘤微血管密度（MVD）。结果：PC-3细胞经DHA处理后,SEM观察到典型的凋亡细胞;裸鼠种植瘤经治疗后,TEM观察肿瘤组织中散在凋亡细胞及凋亡小体,且DHA大小剂量组MVD均低于对照组（P〈0．05）。结论：DHA具有较强的抗肿瘤作用,其作用机制可能与诱导肿瘤细胞凋亡有关。     

汉防己甲素对兔眼角膜基质细胞增殖和凋亡作用的比较研究

目的：探讨汉防己甲素（Tet）对离体兔眼角膜基质细胞抑制增殖作用,及其对增殖率和凋亡率的作用程度比较。方法：选用原代及传代兔角膜基质细胞,实验组加入含不同浓度Tet的培养液,对照组加入含等量PBS的培养液。通过免疫细胞化学技术了解细胞的PCNA表达来枪测细胞增殖情况,计算增殖率并测定变化;流式细胞仪（FCM）测定细胞周期和凋亡率的变化。结果：Tet质量浓度为2μg／ml,3μg/ml,4μg／ml时,作用48h,随着Tet浓度的增大,PCNA阳性细胞数明显减少,角膜基质细胞的增殖率呈递减趋势,存在剂量一效应关系（P〈0．05）;FCM结果显示,随着Tet的浓度增大,实验组G0／G1期细胞增加,细胞删期阻滞在G1期,见细胞凋亡峰,凋亡率也随浓度增大而升高（P〈0．05）;Tet对增殖率影响的曲线斜率绝对值明显大于对调亡率作用的斜率绝对值,结论：Tet对角膜基质细胞的PCNA的表达具有明显抑制作用,随Tet浓度增大,角膜基质细胞的增殖率下降,Tet通过将细胞阻滞在G1期而发挥其抗增殖作用,且存在剂量效应关系;Tet诱导细胞凋亡,且也存在剂量效应关系,随浓度增大,凋亡率增加;在2μg/ml．3μm/ml,4μg／ml的浓度内,Tet对角膜基质细胞同时具有抑制增殖和诱导凋亡作用,但以抑制增殖作用为主.     

低强度超声对鼻咽癌CNE2细胞增值和凋亡的影响

目的：观察低强度超声对人鼻咽癌CNF2细胞增殖和凋亡的影响。方法：以低强度超声（频率1．7MHz,剂量1．35W／cm2）辐照CNF2细胞,采用MTT观察超声对COCl细胞增殖的抑制作用;光镜观察细胞形态,细胞核荧光染色法检测凋亡细胞。结果：超声波能够显著抑制鼻咽癌CNE2细胞增殖,辐照后孵育18h,其MTT光吸收值与对照组相比监著降低：光镜下可见细胞形态学改变,同时Hoechst 33258荧光染色法观察到亮蓝色着染的凋亡细胞。结论：低强度超声辐照对人鼻咽癌CNE2细胞体外生长具有明显的抑制作用,并可诱导其凋亡。     

Ras信号通路阻断剂FTI-277抑制HeLa细胞增殖及对cyclin E表达的影响

目的：观察Ras信号通路阻断剂（FTI-277）在人类宫颈癌细胞株HeLa细胞中对细胞周期素E（cyclin E）表达及对HeLa增殖、凋亡的影响情况。方法：采用MTT法观察不同浓度的FTI-277对HeLa细胞的生长抑制作用;流式细胞术（FCM）观察Hela细胞的细胞周期变化;RT-PCR、Western blot法检测FTI-277阻断Ras信号通路前后HeLa细胞cyclin E mRNA及蛋白表达。结果：各浓度范围FTI-277均能抑制HeLa细胞的增殖,且具有剂量依赖性和时间依赖性。流式细胞术分析显示,FTI-277作用后的HeLa细胞明显滞留于G1/S期,且具有时间依赖性。RT-PCR及Western blot结果显示,FTI-277作用前后HeLa细胞cyclin E mRNA表达无明显变化,而蛋白表达水平明显降低。结论：FTI-277可以抑制细胞增殖,Ras信号转导通路可能是通过影响HeLa细胞cyclin E转录后的翻译环节减少其表达。     

Survivin-siRNA对骨肉瘤MG-63细胞药物敏感性的研究

目的研究特异性Survivin-siRNA转染骨肉瘤MG-63细胞后对骨肉瘤细胞药物敏感性的影响。方法应用两种特异性Survivin-siRNA（Survivin-siRNA,Survivin—siRNA,）转染骨肉瘤MG-63细胞,RT—PCR检测MG-63细胞Survivin-mRNA表达,流式细胞术检测MG-63细胞的凋亡,MTT法检测顺铂、多柔比星对转染前后MG-63细胞的半数抑制浓度（IC50）。结果特异性Survivin-siRNA转染骨肉瘤MG-63细胞24h后SurvivinmRNA表达水平明显抑制;流式细胞术显示转染Survivin-siRNA的MG-63细胞48h后凋亡率明显高于非特异性siRNA组和空白对照组（F=17．32,P〈0．01）;特异性Survivin—siRNA增强了MG-63细胞对多柔比星的敏感性5倍,增强了MG-63细胞对顺铂的敏感性9倍。结论化学合成的Survivin—siRNA能有效抑制Survivin基因在骨肉瘤MG-63细胞中的表达,并提高MG-63细胞对多柔比星、顺铂的敏感性。     

新型光敏剂BF01光动力治疗人肝癌的实验研究

探讨光敏剂BF01光动力对人肝癌细胞株bel- 7402体外杀伤效应和体内抑瘤效 应及其作用机制。CCK-8法检测同一激光强度不同给药浓度的(0、0.8、3.2 μmol·L－1)BF01光动力(激光剂量2.4 J/cm²,光照时间 :1min/well,激光波长652 nm)处 理下对bel-7402细胞的抑制率和IC50,流式 细 胞术检测bel-7402细胞死亡方式,DAPI染色 观察细胞凋亡特征,光动力后检测bel-7402活性氧、共聚焦显微镜 观察亚细胞定位,建立 裸鼠人肝癌细胞模型,绘制肿瘤生长曲线,观察治疗效果。BF01-PDT治疗组能明显 抑制bel-7402肿瘤细胞增殖,BF01的浓度为6.4 μmol·L－1时,其杀伤率到86%,半数杀伤 浓度IC50 μmol·L－1,检测细 胞死亡方式主要是以晚期凋亡为主,BF01-PDT作用 bel-7402细胞后,经DAPI染色细胞核的形态呈现出凋亡的特征,活 性氧水平随给药浓度 增加而增加,亚细胞定位在线粒体,体内实验表明BF01-PDT可以明显抑制人肝癌肿瘤生长 。光敏剂BF01介导的光动力疗法对人肝癌细胞及小鼠体内肿瘤生长抑制作用明显,光 动力疗法以细胞凋亡为主,其机制可能与光敏剂BF01定位在肿瘤细胞的线粒体,增加 bel-7402细胞中ROS含量有关。     

双氢青蒿素体外诱导人乳腺癌细胞凋亡的电镜研究

目的:研究双氢青蒿素(dihydroartemisinin,DHA)在体外对人乳腺癌细胞的抗癌作用。方法:体外培养人乳腺癌细胞株MDA-MB-231,经不同浓度DHA处理后,应用透射电镜观察癌细胞的超微形态学变化。结果:不同浓度DHA处理后的癌细胞,随浓度的增加,呈现典型的细胞凋亡超微结构改变。结论:DHA能诱导乳腺癌MDAMB-231细胞的凋亡,有望成为抗乳腺癌的辅助化疗药物。     

铜蒸气激光照射对血管平滑肌细胞凋亡的诱导作用及对其增殖的影响

目的：研究510.6nm铜蒸气激光照射对体外培养的血管平滑肌细胞（VSMC）凋亡的诱导作用，以及对增殖细胞核抗原（PCNA）表达的影响，探讨铜蒸气激光照射在经皮冠状动脉成形术（PTCA）后再狭窄（RS）的防治作用。方法：贴块法培养兔VSMC，510.6nm铜蒸气激光照射后透射电镜观察凋亡细胞形态学改变，TUNEL法计数凋亡细胞，免疫组化染色法计数照光对PCNA阳性表达率的影响。结果：激光照射后，VSMC凋亡率较未照光组增加12.8倍，而PCNA表达阳性率降低27．9％倍；电镜下观察细胞呈典型的凋亡形态学改变。结论：铜蒸气激光照射可以诱导VSMC凋亡，而且抑制其增殖，在RS的防治中具有一定的作用。     

竹红菌乙素光动力对人舌鳞癌Tca8113细胞杀伤作用的初步研究

目的：本文是用实验的方法,初步探讨中药光敏剂竹红菌乙素（HB）在光动力（PDT）作用下,对体外人舌鳞癌Tca8113细胞的杀伤作用。方法：常规培养、选取对数生长期人舌鳞癌Tca8113细胞,用含有竹红菌乙素（HB）浓度分别为0μM、0．25μM、0．5μ／M、1μM、2μM的RPMI-1640培养液孵育细胞4h后,经470nmLED定制多光源半导体激光光照处理（PDT）,能量密度设置分别为0J／CM^2、1J／CM^2、2J／CM^2、3J／CM^2、4J／CM^2。PDT处理后细胞继续孵育24h后,分别在光学显微镜和荧光显微镜下观察细胞形态学变化,并用甲基噻唑四唑法（MIT法）检测竹红菌乙素（HB）对细胞的抑制作用。并分别在激光照射6h及24h后,用流式细胞仪测定细胞凋亡率。结果：经PDT处理后细胞在光学显微镜及荧光显微镜下均可观察到细胞坏死和凋亡样改变;在一定浓度范围和光照能量密度范围内,竹红菌乙素（HB）在PDT作用下对人舌鳞癌Tca8113细胞生长增殖的抑制和诱导凋亡作用与药物浓度和能量密度成正相关变化。结论：竹红菌乙素（HB）在光动力（PDT）作用下,对人舌鳞癌Tca8113细胞具有显著的杀伤作用,并在一定范围内呈浓度剂量和光剂量正相关依赖性。     

乳腺癌细胞exosomes对人脐静脉内皮作用的实验研究

目的:观察人乳腺癌细胞株MDA-MB-231细胞源exosomes对人脐静脉内皮细胞株(HUVECs)增殖、迁移能力及血管样结构形成的影响.方法:超速离心及密度梯度离心法提取exosomes;MTT法检测MDA-MB-231细胞源exosomes对HUVECs增殖的影响;Transwell小室法检测HUVECs与exo...     

三氧化二砷对照射后人肺癌细胞MGMT和VEGF表达的影响

目的：本文探讨三氧化二砷（Arsenic trioxide,As2O3）对受照后人肺癌A2细胞株MGMT和VEGF表达的影响。方法：设立空白对照组、照射组（直线加速器X射线照射,2Gy）、As2O3组（1.0μmol/L As2O3）、As2O3＋照射组（1.0μmol/L As2O3＋X射线照射,2Gy）。平板克隆形成试验测定各实验组细胞集落形成率,用RT-PCR和免疫组化检测MGMT（甲基鸟嘌呤甲基转移酶）、VEGF（血管内皮生长因子）基因和蛋白水平的变化。结果：As2O3＋照射组细胞集落形成率较As2O3组及照射组明显下降,与As2O3组或照射组比较差异具有显著性（P〈0.01）。As2O3＋照射组细胞MGMT、VEGF基因表达明显减少,而且MGMT、VEGF蛋白的表达明显降低,与As2O3组或照射组比较差异具有显著性（P〈0.01）。结论：As2O3能使受照后人肺癌A2细胞MGMT、VEGF基因及蛋白表达水平明显下调,可能是As2O3对人肺癌细胞辐射增敏的机制之一。     

双氢青蒿素对前列腺癌裸鼠种植瘤CHOP表达的影响

目的：研究双氢青蒿素对前列腺癌PC-3细胞裸鼠种植瘤CHOP表达的情况,并探讨其作用途径及机制。方法：采用人前列腺癌PC-3细胞建立裸鼠种植瘤模型,20只模型鼠随机分为对照组、溶剂组、实验1组及实验2组,经13d干预后,免疫组织化学法检测CHOP的表达水平;透射电子显微镜观察DHA对PC-3细胞种植瘤超微结构的影响。结...     

荧光染色法观察LED活化竹红菌素介导的光动力作用诱导卵巢癌细胞凋亡

目的:探讨LED活化竹红菌素介导的光动力作用诱导人卵巢癌细胞凋亡的光化学生物学效应.方法:应用Hoechst33258细胞核荧光染色法观察LED活化竹红菌素介导的光动力作用对人卵巢癌细胞凋亡的影响,结果:LED活化竹红菌素介导的光动力作用组出现核凝聚、凋亡小体的人卵巢癌细胞株H08910细胞明显多于单纯LED辐照组、单...     

Role of mitochondria in the switch mechanism of the cell death mode from apoptosis to necrosis--studies on rho0 cells

Detailed mechanisms of the switch of the cell death mode from apoptosis to necrosis remain to be solved, although the intracellular level of ATP and that of free radicals have been postulated to be the major factors involved in the mechanisms. In the present study menadione (MEN)-induced cell injury processes were studied using rho0 cells derived from human osteosarcoma 143B cells and parental rho+ cells co-treated with inhibitors of electron transfer chain of mitochondria or oligomycin, an inhibitor of ATP synthesis. Treatment of rho+ cells with 100 microM MEN induced apoptosis, which reached the maximum at 6 h, and was followed by an abrupt decrease thereafter, while necrotic cells (NC) increased continuously when they were judged by Annexin V and PI double staining. On the other hand, MEN induced apoptotic and necrotic changes much faster in rho0 cells compared to rho+ cells. The frequency to find apoptotic cells (AP) in the former cells was distinctly smaller than that to find NC judged by Annexin V and PI double staining. Electron microscopically, a major population of rho0 cells treated with MEN for 6 h consisted of intermediate cells, and a small number of AP co-existed. At 9 h of the treatment intermediate cells were exclusively seen, and AP were hardly detected. When parental rho+ cells were treated with MEN in the presence of oligomycin or oligomycin plus antimycin A both apoptotic and necrotic changes of the cells were distinctly accelerated. The intracellular level of superoxide in rho0 cells continuously increased after the MEN treatment, whereas that of ATP remained distinctly low before and after the MEN treatment compared to that in rho+ cells. These data suggest that the intracellular level of superoxide may be a key factor controlling the switch from apoptosis to necrosis.     

Modification of physicochemical properties of actin filaments suppresses cell fragmentation in the execution phase of staurosporine-induced apoptotic processes

Effects of jasplakinolide (JSP), a stabilizer of F-actin, and latrunculin A (LTA), a destabilizer of F-actin, on a series of events occurring in the execution phase of staurosporine (STS)-induced apoptotic processes were studied using human osteosarcoma 143B cells. Time-dependent apparent increases of the population of cells with collapsed membrane potential of mitochondria (Delta Psi(m)) caused by STS treatment were not due to actual decreases in the Delta Psi(m) per cell, but due to the fragmentation of cells resulting in decreases in the number of active mitochondria per cell. Decreases in the Delta Psi(m) in fragmented cells occurred late in the execution phase. Both JSP and LAT failed to prevent STS-induced release of cytochrome c from mitochondria followed by the activation of caspases 3 and 9, the cleavage of poly (ADP-ribose) polymerase (PARP) and apoptotic nuclear fragmentation. However, both drugs prevented STS-induced apoptotic cell fragmentation and decreases in the Delta Psi(m). These results indicate that physicochemical states of actin filaments play a certain role in the execution phase of STS-induced apoptotic processes.     

趋化因子配体18与鼻咽癌相关性研究及意义

目的：探讨趋化因子配体18（CCL18）在鼻咽癌患者血浆中的表达水平及检测在体外对人鼻咽癌细胞株增殖和迁移能力的影响。方法：收集到重庆医科大学附属第二医院就诊并病理诊断为鼻咽癌患者37例,在未接受任何治疗前留取血液标本,另取20例健康体检者外周血液标本作为正常人群对照。以酶联免疫吸附测定（ELIAS）方法检测血浆中CCL18的表达水平。体外实验以人鼻咽癌细胞株HNE-1为研究对象,用噻唑蓝（MTT）比色法检测重组CCL18对细胞增殖能力的影响,用Transwe11体外迁移系统检测CCL18对鼻咽癌细胞的趋化作用。结果：鼻咽癌患者血浆中CCL18的表达水平明显高于健康体检者（P〈0．0001）,伴有颈部淋巴结转移的鼻咽癌患者较未发生颈部淋巴结转移者血浆中CCL18的表达水平显著升高（P〈0．05）。CCL18因子在体外对鼻咽癌细胞无明显促进增殖作用（P〉0．05）。CCL18对鼻咽癌细胞具有明显促进迁移趋化作用,并呈现浓度依赖性。结论：CCL18是鼻咽癌的生物学标志物,其表达与鼻咽癌的侵袭转移相关,对鼻咽癌的诊断和预后判断有一定临床意义。     

Ultrastructural basis for the transition of cell death mode from apoptosis to necrosis in menadione-treated osteosarcoma 143B cells

Time-dependent ultrastructural changes of menadione-treated human osteosarcoma 143B cells were correlated with those in their stainability to Annexin V and propidium iodide (PI). Populations of both apoptotic (Annexin V(+)/PI(-)) and necrotic (Annexin V(+)/PI(+)) cells, judged by flow cytometry, began to increase at 2 h after menadione treatment. The former reached a maximum at 6 h followed by abrupt decreases thereafter, while the latter continued to increase. Electron microscopically, cells obtained at 6 h after the menadione treatment consisted of mixed populations of cells with typical apoptotic features and those with a mixture of apoptotic and necrotic features, while cells obtained at 8-24 h consisted exclusively of cells with a mixture of apoptotic and necrotic features. Thus, necrotic cells, as judged by flow cytometry, were in a transitional state of cell death mode from apoptosis to necrosis and are thus designated as 'intermediate cells'. Lack of apoptotic bodies, judged by flow cytometric analysis on sub-G1 nuclei and by electron microscopy in menadione-treated cells, suggested that the transition of cell death mode from apoptosis to necrosis occurred before the apoptotic processes were completed. Effects of N-acetylcysteine and Z-VAD-fmk on menadione-induced ultrastructural changes were also studied.