TRAIL诱导骨肉瘤HOS-8603细胞凋亡与线粒体跨膜电位关系的研究

目的 研究TRAIL诱导HOS-8603细胞凋亡与线粒体跨膜电位（ΔΨm）关系。方法 MTT法测定TRAIL对HOS-8603细胞的抑制率; 将HOS-8603细胞分别用TRAIL、TRAIL ＋ 1.0μg/ml 环孢菌素A（CsA）处理, 然后用透射电镜观察HOS-8603细胞形态学变化；用流式细胞术分别测定亚G1期细胞百分率及PI和Rh123双染色后细胞的ΔΨm。 结果 MTT还原试验表明：作用24小时后, 2.5μg/mlTRAIL的抑制率为29%；5μg/mlTRAIL的抑制率为83%. HOS-8603细胞经TRAIL作用后, 透射电镜下观察到典型的凋亡形态特征；随着TRAIL作用时间增加, HOS-8603细胞凋亡数增加和ΔΨm 降低（F=33.831 P〈0.01） , 两者呈直线相关.CsA不仅能抑制TRAIL诱导HOS-8603细胞ΔΨm 降低。而且能减少凋亡细胞数的增加（t=7.103 ,P〈0.01）。结论 TRAIL诱导HOS-8603细胞凋亡的线粒体依赖途径是通过使线粒体膜通透性转运孔开放, ΔΨm降低来实现；CsA能部分抑制这些效应。     

顺铂诱导骨肉瘤HOS-8603细胞凋亡并降低其线粒体跨膜电位

目的研究顺铂诱导HOS-8603细胞凋亡与线粒体跨膜电位(ΔΨm)关系。方法MTT法测定顺铂对HOS-8603细胞的抑制率;将HOS-8603细胞分别用顺铂、顺铂+1.0ug/ml环孢菌素A(CsA)处理,用透射电镜观察HOS-8603细胞形态学变化;用流式细胞术分别测定亚G1期细胞百分率及PI和Rh123双染色后细胞的ΔΨm。结果MTT还原试验表明,作用24小时后,1ug/ml和50ug/ml顺铂的抑制率分别为58.56%,90.69%;HOS-8603细胞经顺铂作用后,透射电镜下观察到典型的凋亡形态特征;随着顺铂作用时间增加,HOS-8603细胞凋亡数增加和ΔΨm降低(P<0.01),两者呈直线相关,CsA能部分抑制这些效应。结论顺铂诱导HOS-8603细胞凋亡的另一途径是通过使线粒体膜通透性转运孔开放,ΔΨm降低来实现的。     

塞来昔布抑制人骨肉瘤细胞的研究

目的　研究塞来昔布(celecoxib)对人骨肉瘤细胞株HOS-8603生长和凋亡的影响,并探讨其作用机制。方法　采用四唑盐(MTT)比色法检测细胞增殖,以确定塞来昔布对人骨肉瘤细胞株HOS-8603的有效剂量;应用高效液相色谱(HPLC)检测前列腺素E2(PGE2 )含量;光镜形态学观察,流式细胞仪测定细胞周期。结果　塞来昔布抑制人骨肉瘤细胞株HOS-8603生长和诱导凋亡(当celecoxib的浓度为80umol/L时抑制率达到46. 75 ±7. 697% );这种抗增殖作用可能与celecoxib对COX-2的抑制作用有关;且不能被PGE2所拮抗。塞来昔布能够诱导HOS-8603细胞凋亡,当其浓度为80umol/L时,凋亡率增加了44. 7% (46. 0%比1. 3% );但是塞来昔布的这种抑制HOS-8603细胞生长和诱导凋亡作用不能被PGE2 所拮抗。结论　塞来昔布对于人类骨肉瘤可能是一种有效的化学治疗和化学预防药物,塞来昔布并非通过前列腺素E2(PGE2 )途径抑制人骨肉瘤细胞株HOS-8603生长和诱导凋亡。     

新型光敏剂CPD4对人乳腺癌细胞体外光动力杀伤效应

目的:研究新型光敏剂叶绿素衍生物4(chlorophyll derivative,CPD4)对人乳腺癌细胞系T47D和MCF-7体外光动力杀伤效应,并对机理进行初步探讨.方法:采用激光共聚焦荧光显微镜结合荧光探针标记技术观察光敏剂在细胞内分布及亚细胞定位;将终浓度分别为2.5、2.0、1.5、1.0、0.5 μg/mL的CPD4加入到T47D及MCF-7细胞培养皿中,孵育2h后用670nm半导体激光治疗仪照射,光剂量为10J/cm2,照毕,继续培养24h后采用染料排斥实验测定细胞的存活率.流式细胞仪Annexin V-FITC/PI双染法检测细胞死亡方式和凋亡率.实验分为四组:空白对照组、单纯光敏剂组、单纯细胞光照组,光敏剂-细胞光照组.结果:光敏剂CPD4可穿过T47D及MCF-7细胞膜进入细胞,分布在细胞膜及胞浆中,线粒体是其亚细胞分布的主要位点之一.光动力组中光敏剂浓度分别为2.5、2.0、1.5、1.0、0.5μg/mL,T47D及MCF-7细胞死亡率分别为(76.77±6.41)%、(40.51±7.28)%、(38.4±5.35)%、(17.08±0.52)%、(11.16±0.28)%、(81.67±1.53)%、(69.67±4.51)%、(45.33±4.73)%、(17.67±2.52)%、(12.00±2.00)%.Annexin V-FITC/PI法检测PDT作用24h后凋亡率分别可达36.80%及40.90%.结论:新型光敏剂CPD4分布在人乳腺癌细胞T47D及MCF-7的细胞膜及细胞浆中,线粒体是其亚细胞分布主要位点之一;光敏剂CPD4对人乳腺癌细胞T47D和MCF-7具有良好的光动力杀伤效应,呈现量效关系,诱导细胞凋亡是其主要杀伤方式.线粒体途径促进细胞凋亡可能是其光动力杀伤机制.     

骨肉瘤特异性细胞毒T淋巴细胞的诱导及研究

本研究探讨骨肉瘤特异性细胞毒T淋巴细胞(OSS-CTL)的诱导方法及其抗瘤特性和杀伤机制.通过生化方法从HOS-8603人骨肉瘤细胞系中提取、纯化、鉴定骨肉瘤相关抗原(OSAA66),然后与低剂量IL-2(100U/ml),CD3(10μg/ml)单折协同刺激骨肉瘤致敏的外周血淋巴细胞,诱导产生OSS-CTL.检测其细胞表型、杀伤机制、体内外的增值能力及抗瘤活性,并与骨肉瘤TIL进行了比较.①流式细胞仪检测OSS-CTL的表型特征为CD3~ (87.6±6.3)%,CD4~ (21.7±4.1)%,CD8~ (94.7±5.3)%,CD11b~ (1.9±0.9)%,HLA-DR~ (79.3±8.7)%,即以CD3~ CD8~ CTL为主异质细胞群.②[3~H]-TdR释放法测定细胞毒性,结果OSS-CTL对HOS-8603和自体骨肉瘤细胞的杀伤活性分别为97.3%和95.2%,而对K562和SHG-44细胞仅为11.2%和9.6%,两组差异显著(P<0.05).提示,OSS-CTL对OSAA66有关的骨肉瘤细胞具有高效的特异杀伤活性.杀伤机制研究表明,在光镜和电镜下观察到OSS-CTL通过接触溶解和诱导凋亡途径杀伤靶细胞.通过流式细胞仪检测发现,效靶比为1:1,50:1,     

ALA-PDT诱导白血病细胞株HL-60凋亡

背景与目的:基于5-氨基乙酰丙酸的光动力疗法(ALA-PDT)利用肿瘤细胞对ALA产生的内源性光敏剂原卟啉IX(PpIX)的优先摄取,使肿瘤细胞在受到一定的光照后被选择性地杀伤。到目前为止,ALA-PDT引起肿瘤细胞破坏的确切机制尚未完全阐明。本研究探讨ALA-PDT对白血病细胞株HL-60的凋亡诱导作用。方法:以白血病细胞株HL-60为实验模型。实验分为4组,对照组、单纯ALA组、单纯光照组及ALA PDT组。用MTT法检测细胞的存活率,瑞氏染色观察细胞形态学改变,用Annexin V-FITC/PI双染法检测细胞凋亡率,并用共聚焦激光显微镜(LSCM)观察凋亡细胞的特征。结果:ALA PDT组光照后细胞形态学可见凋亡改变;MTT法显示细胞存活率明显下降,24 h为(46±9)%,48 h为(26±8)%,两者比较差异有显著性(P<0.05);流式细胞仪检测显示光照后4、5和24 h细胞凋亡率分别为(26±9)%、(29±11)%和(51±6)%,与对照组相比差异有显著性(P<0.05);LSCM观察AnnexinV-FITC单阳性及AnnexinV-FITC/PI双阳性细胞均具有典型的凋亡特征,而单纯ALA组、单纯光照组及对照组则无上述改变。结论:ALA-PDT能杀伤白血病细胞株HL-60,主要通过诱导凋亡的方式实现的,并呈一定的时间依赖性。     

新型靶向光敏剂光动力诱导HeLa细胞凋亡的实验研究

[目的]研究新型靶向光敏剂Ⅰ光动力诱导HeLa细胞死亡的方式。[方法]用流式细胞仪分析照射剂量和光敏剂浓度对HeLa细胞凋亡的影响,细胞凋亡和坏死的比例;并采用激光共聚焦观察细胞F-actin和核的变化;透射电镜观察细胞超微结构的改变。[结果]光敏剂Ⅰ介导的光动力治疗可诱导HeLa细胞的凋亡和坏死,但凋亡的比例明显大于坏死（P=0.0053）,且细胞凋亡的量分别与光照剂量（r=0.987,P〈0.001）和光敏剂浓度（r=0.995,P〈0.001）呈正相关;光动力诱导后,细胞的F-actin结构破坏,细胞皱缩,透射电镜下可见凋亡小体的形成。[结论]光敏剂Ⅰ介导的光动力治疗主要通过凋亡途径导致HeLa细胞的死亡。     

顺铂诱导原代培养人膀胱癌细胞的凋亡

目的：探讨顺铂诱导原代培养膀胱癌细胞发生调亡变化的特征及机制。方法：以0-100umol/L浓度顺铂处理原代培养膀胱癌细胞12-72小时,应用MTT法检测细胞增殖抑制变化。分析其与凋亡指数（AI）的关系;用光镜和透射电镜分别观察膀胱癌细胞凋亡变化的特征。结果：顺铂能够明显地诱导膀胱癌细胞凋亡,具有一定范围内的作用时间和药物浓度依赖性,与细胞增殖抑制密切相关。膀胱癌细胞凋亡的镜下特征为胞膜完整,胞浆浓缩,核碎裂、萎缩或边积,胞浆外突形成凋亡小体。结论：证实顺多功能铂诱导膀胱癌细胞广泛性凋亡是它产生杀瘤效应的途径之一。     

树突状细胞诱导抗人肝癌细胞系的肿瘤免疫

 目的 以树突状细胞(DC)在体外诱导抗肝癌免疫.方法 自肝癌患者外周血中分离DC;以粒/巨噬细胞集落刺激因子(GM-CSF)及白介素-4(IL-4)联合刺激DC;以人肝癌细胞系HepG2细胞和BEL-7402细胞的肿瘤相关抗原(TAA)激活DC;DC诱导自体T淋巴细胞增殖、分化为细胞毒素性T细胞(CTL);检测CTL对HepG2细胞、BEL-7402细胞、SGC-7901细胞、LOVO细胞及HOS-8603细胞的细胞毒作用.结果 肝癌患者外周血DC能够诱导自体T淋巴细胞增殖分化为CTL,该CTL对HepG2细胞和BEL-7402细胞有强大的杀伤力(杀伤率分别为90%±10%,86%±11%),对SGC-7901细胞、LOVO细胞及HOS-8603细胞则无明显的细胞毒作用(杀伤率分别为11%±6%,8%±4%,6%±4%).结论 肝癌患者外周血DC体外能够诱导高效而特异抗肝癌免疫.提示DC可能在治疗肝癌及预防肝癌术后复发和转移中发挥重要作用.     

羟基喜树碱对SW1990体外增殖及凋亡的影响

目的：探讨羟基喜树碱（HCPT）体外抑制人胰腺癌细胞株SW1990增殖并诱导其凋亡的作用。方法：体外培养的胰腺细胞以不同浓度的HCPT处理20至24h后,用MTT法测定细胞增殖,以Annexin V早期凋亡检测试剂盒,电子显微镜,流式细胞仪和原位末端标记分析以及bcl-2免疫细胞化学标记分析检测细胞凋亡情况,结果：MTT细胞增殖,在浓度为3．125ug/ml-100ug/ml各组,药物作用24h,后,肿瘤细胞的增殖抑制率显著高于对照线。（2）原位末端标记及流式细胞仪检测,在0．05mg./ml浓度下,作用20h后,肿瘤细胞的凋亡率在13．2－15．3％之间,在0．1mg/ml浓度下,作用20h后,凋亡率在26．1－30．4％之间。（3）电镜及Annexin V荧光检测：在0．05mg/ml浓度下,凋亡以早期表现为主,早期凋亡率约为15％,（4）bcl-2免疫细胞化学表达：经0．05mg/ml浓度作用24h后,bcl-2表达较对照组明显减少,结论：HCT在体外可抑制SW1990细胞的增殖,部分作用机制是诱导细胞凋亡,可作为细胞凋亡诱导剂用于胰腺癌治疗。     

Repression of telomerase activity during in vitro differentiation of osteosarcoma cells

In this report, we studied the relationship between telomerase activity and in vitro differentiation of osteosarcoma cells. Human osteosarcoma cells (HOS-8603) were treated with all-trans-retinoic acid (RA) and dexamethasone (DEX). Cell cycle phase, alkaline phosphatase (AP) activity, telomerase activity, and human telomerase RNA (hTR) in treated cells were detected. The results showed that the treated cells underwent morphologic differentiation. AP activity of the cells increased significantly. The proportion of the cells in S and G2/M phases was increased. A pronounced decline in telomerase activity was observed, but no significant difference in the amount of hTR expressed, when compared with the control. This study demonstrates that: (1) both RA and DEX can inhibit cell growth and induce morphologic and functional differentiation of HOS-8603 cells; (2) telomerase is an enzyme system regulated during induced differentiation of HOS-8603 cells; (3) significantly decreased telomerase activity may be an indicator of differentiation but does not parallel the expression level of hTR; and (4) the regulation of telomerase is directly linked to cell differentiation not cell cycle.     

Development of meta-tetrahydroxyphenylchlorin-monoclonal antibody conjugates for photoimmunotherapy

A limitation of photodynamic therapy is the lack of tumor selectivity of the photosensitizer. To overcome this problem, a protocol was developed for coupling of meta-tetrahydroxyphenylchlorin (mTHPC), one of the most promising photosensitizers, to tumor-selective monoclonal antibodies (MAbs). mTHPC was radiolabeled with 131I to facilitate the assessment of the in vitro and in vivo behavior. After the modification to 131I-mTHPC-(CH2COOH)4, thus increasing the water solubility and creating a functional moiety suitable for coupling, conjugation was performed using a labile ester. Insoluble aggregates were not formed when mTHPC-MAb conjugates with a molar ratio of up to 4 were prepared. These conjugates showed a minimal impairment of the integrity on SDS-PAGE, full stability in serum in vitro, and an optimal immunoreactivity. To test the in vivo behavior of the mTHPC-MAb conjugates, the head and neck squamous cell carcinoma-selective chimeric MAb U36 was used in head and neck squamous cell carcinoma-bearing nude mice. Biodistribution data showed that the tumor selectivity of cMAb U36-conjugated mTHPC was increased in comparison with free mTHPC, despite the fact that conjugates with a higher mTHPC:MAb ratio were more rapidly cleared from the blood. Preliminary results on the in vitro efficacy of photodynamic therapy with MAb-conjugated mTHPC showed that mTHPC coupled to the internalizing murine MAb 425 exhibited more phototoxicity than when coupled to the noninternalizing chimeric MAb U36.     

Photodynamic action of meta-tetrahydroxyphenylchlorin (mTHPC) on an ovarian cancer cell line

Meta-tetrahydroxyphenylchlorin (mTHPC) exhibits significant cytotoxicity against a variety of human cells in culture in combination with light, but also in dark reaction. The ovarian cancer cell line SK-OV3 was incubated with various concentrations of mTHPC and in comparison with Taxol and Cisplatin: then the effect on cell growth was determined. mTHPC exhibited an IC50 of 0.9 muM after 24 hours incubation (IC50 of 1.25 after 2 hours), whereas Cisplatin and Taxol, which, have been used as first line agents for the treatment of ovarian carcinomas, inhibited cell proliferation with an IC50 concentration of 4.6 muM and 78 nM after 24 hours incubation, respectively. Incubation of SK-OV3 cells with mTHPC for 5 days resulted in cytostatic cytotoxicity at a concentration of 0.5 muM. The photodynamic effect of mTHPC depends/among other parameters/on the concentration of the dye present. In combination with light (approximately 15 J/cm2) a linear relationship between the dose of mTHPC and the amount of necrotic cells was observable. Higher concentrations of mTHPC caused necrosis of the ovarian tumor cells. The intracellular concentration of mTHPC showed a linear increase up to 28.6 nM (incubation concentration). In summary, these studies demonstrated that mTHPC exhibits potent antiproliferative activity by inducing necrosis after application of light. MTHPC might be a promising agent with cytostatic and photodynamic properties for the treatment of metastasing ovarian carcinomas. A sensitive PCR method was not able to show the induction of apoptosis in the SK-OV3 ovarian cell line. Using propidium staining, it could be proved that the cell death was caused by necrosis and not through apoptosis after irradiation with light.     

Evaluation of a new photosensitizer,meso-tetra-hydroxyphenyl-chlorin,for use in photodynamic therapy: A comparison of its photobiological properties with those of two other photosensitizers

The properties of a new photosensitizer, meso-tetra-hydroxyphenyl-chlorin (mTHPC), were studied using V79 cells (Chinese-hamster lung fibroblasts). Comparisons were made with 2 other photosensitizers: photofrin II (PII) and meso-tetra-hydroxyphenyl-porphyrin (mTHPP). A main advantage of mTHPC is that it has a strong absorption at 652 nm. Maximal cellular uptake of the dye was observed after 24 hr incubation of the cells with the drug. Using a confocal laser-scanning fluorescence microscope, we observed a diffuse distribution of m THPC in the cytoplasm. Furthermore, the lipophilicity of mTHPC was compared with that of the components of PII by means of high-pressure liquid chromatography (HPLC). Absorption and fluorescence spectroscopy indicated that aggregated as well as monomeric mTHPC was bound to the cells. The action spectrum for photo-inactivation of the cells showed that aggregated mTHPC did not contribute significantly to its photosensitizing effects. In the present cellular system, the efficiency of photodynamic therapy (PDT) with mTHPC (cells were irradiated at a wavelength of 652 nm) was higher than with PII (irradiation at 630 nm) or with mTHPP (648 nm). The quantum yield for photo-inactivation of cells was smaller for mTHPC than for mTHPP and PII. The addition of 1,3-diphenylisobenzofuran (DPBF) reduced cell inactivation during PDT. Thus, PDT with mTHPC seems to act at least partly via a type-II process. © 1994 Wiley-Liss, Inc.     

Intracellular localization and concentration as well as photodynamic effects of benzoporphyrin derivative monoacid ring A in four types of rodent tumor cells

The relative sensitivities of different tumor cells to photodynamic therapy (PDT) with benzoporphyrin derivative monoacid ring A (BPD-MA) were compared in the four tumor cells. A good correlation was observed between the cell survival at 0.1 microg/ml of BPD-MA and sensitizer uptake/10(6) cells (r = -0.99) or the plating efficiency of cells (r = 0.99). At 3 h after the irradiation, a significant difference was observed in the proportion of apoptotic cells among the four tumor cells (p = 0.024). In conclusion, cell responses to PDT depend on the several factors such as the cell line, photosensitizer dose, and fluence.     

光敏剂mTHPC诱导骨肿瘤细胞凋亡的机制

目的 研究光敏剂对体外培养的人骨肉瘤HOS细胞的作用。方法 在HOS细胞的培养液中加入光敏剂,并孵育一段时间,经光照发挥其光敏作用。结果 经光敏作用后,HOS细胞出现了凋亡现象,表现为荧光染色出现凋亡小体及琼脂糖凝胶电泳见到特征性的梯度条带。结论 光敏剂有诱导HOS细胞凋亡作用。     

Apoptosis induced in vivo by photodynamic therapy in normal brain and intracranial tumour tissue

The apoptotic response of normal brain and intracranial VX2 tumour following photodynamic therapy (PDT) mediated by 5 different photosensitizers (Photofrin, 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX), chloroaluminium phthalocyanine (AlCIPc), Tin Ethyl Etiopurpurin (SnET(2)), and meta -tetra(hydroxyphenyl)chlorin (m THPC)) was evaluated following a previous analysis which investigated the necrotic tissue response to PDT at 24 h post treatment. Free DNA ends, produced by internucleosomal DNA cleavage in apoptotic cells, were stained using a TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling) assay. Confocal laser scanning microscopy (CLSM) was used to quantify the local incidence of apoptosis and determine its spatial distribution throughout the brain. The incidence of apoptosis was confirmed by histopathology, which demonstrated cell shrinkage, pyknosis and karyorrhexis. At 24 h post PDT, AlClPc did not cause any detectable apoptosis, while the other photosensitizers produced varying numbers of apoptotic cells near the region of coagulative necrosis. The apoptotic response did not appear to be related to photosensitizer dose. These results suggest that at this time point, a minimal and fairly localized apoptotic effect is produced in brain tissues, the extent of which depends largely on the particular photosensitizer.     

5-氨基酮戊酸光动力疗法对CNE细胞迁移和侵袭的影响

 【摘要】　目的　观察5-氨基酮戊酸光动力疗法（5-ALA-PDT）作用于人类鼻咽癌细胞株CNE细胞后细胞生长抑制、迁移和侵袭性的改变。方法　通过MTT法检测5-ALA-PDT CNE细胞后,不同参数（5-ALA浓度及激光器能量密度）下的细胞生长抑制率变化。并观察不同参数下5-ALA-PDT后细胞划痕实验及Transwell侵袭小室试验。结果　5-ALA-PDT能明显抑制鼻咽癌高分化CNE细胞株的生长。5-ALA-PDT后,当药物浓度和能量密度达到一定水平时CNE细胞的迁移被抑制。并且药物浓度在饱和量1 mmol／L时,细胞迁移距离与光照时间呈线性负相关（P＜0.05）。光动力作用后,CNE细胞的侵袭能力也被抑制,与5-ALA浓度及激光的能量密度相关。在浓度相同的情况下,细胞侵袭数与照光时间呈线性负相关（r＞0.8,P＜0.0001）。结论　5-ALA-PDT作用于CNE细胞后,细胞的迁移和侵袭能力被抑制,抑制作用与能量密度和药物浓度相关。     

Cellular uptake, subcellular localization and photodamaging effect of temoporfin (mTHPC) in nasopharyngeal carcinoma cells: comparison with hematoporphyrin derivative

Temoporfin (meta-tetra (hydroxyphenyl)chlorin; mTHPC) potentiated a 100-fold higher cytotoxic effect than hematoporphyrin derivative (HPD) on two nasopharyngeal carcinoma cell lines (HK1 and CNE2) in terms of the overall photodynamic therapy (PDT) dose. The cellular uptake, evaluated by flow cytometry and spectrophotometry demonstrated that mTHPC exhibited higher uptake ability than HPD. Confocal laser scanning microscopy detection for both the sensitizer and mitochondria probe on the same cell images revealed that both drugs accumulated diffusely in the cytoplasm and that mitochrondria is a target organelle. Photo-activation ruptured the mitochrondria, with more pronounced mitochondrial damage being observed in mTHPC-PDT course. This correlated well with the cell photokilling efficiency of mTHPC.