平阳霉素对人脐静脉内皮细胞系ECV304的抑制作用及其机制的实验研究

目的：探讨平阳霉素对人脐静脉内皮细胞系ECV304的抑制作用及其相关机制。方法：用不同浓度的平阳霉素作用ECV304细胞不同时间,以MTT法比较平阳霉素作用24、48及72h后的细胞抑制率,应用DNA电泳实验、流式细胞术分析细胞凋亡、坏死情况、细胞周期及Fas、Bcl-2蛋白的表达。结果：MTT法显示随平阳霉素浓度升高和作用时间的延长,细胞生长抑制越明显DNA电泳证实合适浓度平阳霉素作用下细胞发生凋亡,过高浓度细胞则发生坏死;流式细胞术显示细胞凋亡率和坏死率随药物浓度和作用时间增加而增高,过高浓度细胞坏死占主导;平阳霉素各浓度作用24h后,G2—M期百分率增高,S期百分率降低,G0—G1期百分率无明显变化;Fas表达随药物浓度增加而递减,Bcl-2表达随药物浓度增加而升高。结论：平阳霉素能明显抑制ECV304细胞生长,并具有浓度和时间依赖性;平阳霉素可能通过诱导凋亡和坏死作用以抑制ECV304细胞的体外生长和增殖;细胞凋亡发生在G2—M期,是通过上调Fas蛋白并下调Bcl-2蛋白的表达来实现的。     

平阳霉素抑制舌鳞癌细胞系的量效和时效关系及可能的抗癌机制

目的:探讨平阳霉素体外抑制口腔鳞癌细胞系的量效和时效关系及可能的抗癌机制。方法:用不同浓度的平阳霉素培养人舌鳞癌细胞系Tca8113不同时间,以MTT法和荧光显微镜观察细胞增殖抑制情况,流式细胞术分析细胞凋亡率。结果:MTT法和荧光观察显示:不同浓度平阳霉素组间细胞生长抑制率有显著差异(P<0.05),浓度越高,抗增殖效应越明显;随药物作用时间延长,肿瘤细胞生长受抑制递增(P<0.05)。流式细胞术显示:凋亡率随药物浓度增加而增加,但与作用时间长短无关。低浓度药物无法诱导细胞凋亡。结论:平阳霉素在体外能明显抑制人舌鳞癌细胞系Tca8113生长,并具有浓度和时间依赖性。其抗癌机制可能包括细胞毒作用和启动程序化死亡信号等。     

热化疗诱导舌鳞癌CAL-27细胞免疫原性死亡的实验研究

目的:探讨平阳霉素（pingyangmycin, PYM）、热疗（hyperthermia, HT）及两者联合能否诱导舌鳞癌CAL-27细胞发生免疫原性死亡。方法:对人舌鳞癌细胞CAL-27分别进行PYM、HT及两者联合处理,采用CCK-8实验、Annexin V/PI联合染色、流式细胞术及酶联免疫吸附实验探讨不同处理方式对细胞增殖、凋亡、CRT膜表达及HMGB1分泌的影响。采用SPSS 20.0软件包对数据进行统计学分析。结果:PYM抑制CAL-27细胞活性,且呈浓度依赖性（P<0.05）。PYM及HT均使CRT膜表达率升高;两者联合应用,细胞凋亡、CRT膜表达率及HMGB1分泌均较未处理组、单纯化疗组及单纯HT组升高,差异有统计学意义（P<0.05）。结论:PYM化疗及HT均能诱导舌鳞癌CAL-27细胞CRT由胞内至膜表面转位,并促进HMGB1分泌;两者联合应用,无论在诱导凋亡还是在诱导免疫原性死亡方面,效果优于单纯PYM化疗组。     

Experimental study of the effect of TNP-470 on human salivary adenoid cystic carcinoma cell in vitro]

OBJECTIVE: To investigate the effects of TNP-470 on human salivary adenoid cystic carcinoma cell line ACC M in vitro. METHODS: Cell proliferation was assessed by MTT assays and dye exclusion counting. Morphological changes of apoptosis were observed with fluorescent microscope. DNA ladder, apoptosis rate and cell cycle were examined by DNA agarose gel electronphores and fluorescence flow cytometry (FCM), respectively. RESULTS: The 50% inhibitory concentrations (IC50) of TNP-470 on ACC-M cells proliferation by MTT assays and dye exclusion counting were 40.68microg/ml and 46.38microg/ml. Apoptosis were observed by fluorescent microscope. DNA electrophoresis for the cells treated with TNP-470 showed brighter DNA ladder; Sub-G1 peak and G2/M arrest were also determined by FCM (P<0.01). CONCLUSION: TNP-470 has the effect of inducing apoptosis in ACC-M cells in vitro, which may be one of its antitumor mechanisms.     

平阳霉素引发细胞凋亡与MAPK信号转导通路的关联性

目的 探讨平阳霉素引发细胞凋亡与分裂原激活的蛋白激酶家族（mitogen-activated protein kinase,MAPK)信号转导通路的关联性。方法 采用Western印迹法（western blot)检测分裂原激活蛋白激酶的表达。结果 平阳霉素作用KB细胞，随作用时间延长或药物浓度增加，磷酸化细胞外信号调节激酶的表达越不明显；而p-38MAPK蛋白激酶的表达无明显变化。结论 平阳霉素引发细胞凋亡与分裂原激活的蛋白激酶家族信号转导通路有关。     

绞股蓝皂甙及平阳霉素对口腔鳞癌颈淋巴结转移癌细胞抑制作用的研究

目的 :探讨绞股蓝皂甙 (gypenosides,GP)及平阳霉素 (PYM )对口腔鳞癌颈淋巴结转移癌GNM细胞的体外抑制作用及作用机制。方法 :采用MTT法及流式细胞仪进行分析。结果 :GP与PYM对GNM细胞均有抑制作用 ,抑制率与药物浓度呈正相关 ,二者联用 ,抑制率增强。GP主要作用于细胞的“S”期。结论 :GP与PYM对GNM细胞具有抑制作用 ,GP对细胞的损伤是通过干扰细胞周期实现的 ,GP与PYM联用抑制作用增强在于二者作用的细胞周期不同。提示用药时可根据二者各时相时间分开应用。GP具有临床应用价值。     

苦参碱对KB细胞及其多药耐药细胞KBv200的凋亡诱导作用

目的　观察中药苦参的提取物苦参碱对口腔上皮癌KB细胞及其多药耐药细胞KBv200的凋亡诱导作用。 方法　MTT法观察苦参碱对体外培养的KB及KBv200细胞存活率的影响。吖啶橙/溴乙啶(AO/EB)双荧光染色检 测二者细胞凋亡率。流式细胞术分析苦参碱对细胞周期的影响;扫描电镜观察苦参碱作用后的细胞形态学变化。 结果　苦参碱质量浓度在0·50、1·00、1·50、2·00 mg/ml时,均有抑制KB及KBv200增殖的作用,双荧光染色及流式细 胞术提示苦参碱可诱导KB及KBv200细胞凋亡,使细胞生长停滞在S期;扫描电镜观察发现不同质量浓度的苦参 碱作用24 h后,细胞出现体积缩小、细胞质空泡化、细胞核碎裂等凋亡现象。结论　苦参碱对KB及KBv200细胞有 增殖抑制作用,可诱导KB及KBv200细胞凋亡,其机制可能与其导致细胞S期阻滞有关。     

谷胱甘肽及其酶系统与抗癌药物的相互作用

目的：本研究探讨国产抗癌药平阳霉素作用培养肿瘤细胞时与胞内谷胱肽及其酶系统的关联性，改变谷胱甘肽的含量对平阳霉素药效的影响。方法：采用高效能液相层析仪分析法及MTT比色试验法进行检测，结果：平阳霉素可通过谷胱甘肽及其酶系统失活；改变细胞内谷胱甘肽的含量可以逆转平阳霉素的毒性作用，结论：平阳霉素的毒性作用与谷胱甘肽及其酶系统有密切相关。     

双硫仑联合铜通过调控视神经萎缩蛋白1诱导舌癌细胞凋亡

目的 探讨视神经萎缩蛋白1（OPA1）在双硫仑联合铜（DSF-Cu）诱导舌癌细胞凋亡中的作用。 方法 用不同浓度的DSF-Cu干预舌癌细胞Cal-27，MTT法检测细胞增殖活性，Annexin V-FITC/PI染色结合流式细胞仪检测细胞凋亡，荧光素酶发光法检测ATP含量，Western blot法检测OPA1蛋白表达水平。 结果 DSF-Cu能抑制舌癌细胞的增殖活性，促进细胞凋亡的发生，减少细胞ATP含量，并呈浓度依赖性。Western blot结果示低浓度DSF-Cu对OPA1蛋白表达无明显影响，而中、高浓度的DSF-Cu显著抑制OPA1蛋白表达水平。 结论 DSF-Cu可诱导舌癌细胞凋亡，其机制可能是通过抑制OPA1表达。     

Involvement of oxidative stress in mutagenicity and apoptosis caused by dental resin monomers in cell cultures

OBJECTIVE: This investigation studied the possibility that apoptosis as well as mutagenicity induced by resin monomers are mediated by oxidative stress. METHODS: A range of dilutions of three resin monomers (GMA, TEGDMA, and HEMA) was added to culture medium (DMEM/10% FBS), of V79-4 fibroblasts and RPC-C2A pulp cells for 24 h. Their cytotoxic effects were measured by a colorimetric functional assay (MTT). Chromosomal aberration induced by the resin monomers was investigated by counting micronuclei in V79-4 cells. The effects of the resin monomers on DNA fragmentation were viewed by agarose gel electrophoresis of DNA, isolated from RPC-C2A pulp cells that were treated by resin compounds. Resin monomer-induced apoptosis was further confirmed by flow cytometry (staining with both annexin V-FITC and PI). RESULTS: All monomers exhibited a dose-dependent cytotoxic effect, and the ranking of the cytotoxicity based on TC50 was GMA > TEGDMA > HEMA. The resin monomer-induced cytotoxicity was significantly decreased by co-treatment with N-acetylcystein (NAC), an antioxidant. The authors also confirmed a dose-dependent genotoxicity of the resin monomers that had induced micronucleated cells in V79-4 fibroblasts. Similar to the effects on cytotoxicity, NAC reduced the numbers of micronuclei in comparison with those generated by the resin monomers. The preventive effects of NAC were also observed in monomer-induced apoptosis in RPC-C2A cells. A DNA ladder pattern, characteristic of apoptosis, was shown at cytotoxic concentrations, but NAC blocked the resin monomer-mediated DNA fragmentation. The preventive effects of NAC on apoptosis were confirmed by Annexin V staining. Cells exposed to 300 microM GMA, 7 mM TEGDMA, or 14 mM HEMA for 24 h showed a significant increase in apoptotic cells, while NAC co-treatment caused a reduction in apoptotic cells compared to controls. SIGNIFICANCE: These findings suggest that glutathione depletion and oxidative stress are responsible for GMA, TEGDMA, and HEMA-induced mutagenicity and apoptosis.     

Effect of chlorophyllin on normothermic storage of human periodontal ligament cells

The purpose of the present study was to evaluate whether chlorophyllin could serve as an effective constituent of a storage medium to enhance the human periodontal ligament (PDL) cell viability.Freshly isolated PDL cells from premolars extracted from healthy people were stored at 37 degrees C for 6 h in various solutions: F-medium and Hank's balanced salt solution (HBSS), supplemented with chlorophyllin. From MTT viability assays, the highest cell viability was found in the PDL cells stored in HBSS supplemented with 500 nM chlorophyllin, and the chlorophyllin-treated cells showed a dose-dependent response to concentration. Additionally, the results from flow cytometry showed that 77 to 80% of the PDL cells were in the G0/G1 phases of the cell cycle, which suggested that most were in a stable stage.These result showed that HBSS, supplemented with chlorophyllin, may be a useful solution for preserving the viability of PDL cells.     

Plasma-arc generated light inhibits proliferation and induces apoptosis of human gingival fibroblasts in a dose-dependent manner.

OBJECTIVE: This study examined the effects of blue light exposure on the proliferation and cytotoxicity of human gingival fibroblasts (HGF). Cellular mechanism by which blue light causes cytotoxic effects was also investigated. METHODS: HGF were exposed to the plasma-arc generated blue light with various energy densities ranging from 2 to 48J/cm(2). After light exposure of the cells, they were processed for analyzing tritium incorporation, succinate dehydrogenase (SDH) activity, trypan blue exclusion, and DNA fragmentation. In addition, possible mechanism of the light-mediated cytotoxicity was investigated through flow cytometric and Western blot analyses. RESULTS: Blue light exposure significantly inhibited proliferation and SDH activity of HGF in a dose-dependent manner; exposure more than 12J/cm(2) had a toxic effect on the cells. The blue light-induced cytotoxicity of the cells resulted from apoptosis, as proven by the migration of many cells to the sub-G(1) phase of cell cycle and the appearance of DNA ladders. Additional experiments revealed that blue light induces apoptosis of HGF through mitochondrial stress and poly (ADP ribose) polymerase cleavage. SIGNIFICANCE: This study suggests that plasma-arc generated blue light exerts some harm to cells, particularly damaging effect to DNA, and thus a long curing time more than recommended can cause biological damage on the oral tissue.     

An in vitro evaluation of the growth of human periodontal ligament fibroblasts after exposure to a methacrylate-based endodontic sealer

The cytotoxicity of Epiphany root canal sealer at various concentrations from 25-800 microg/mL on human periodontal ligament (HPDL) fibroblasts was evaluated at 1, 3, and 7 days. Controls included untreated cells and cells treated with the vehicle for Epiphany suspension. Fibroblast viability was assessed by 2 methods, crystal violet staining in 24-well plates and the fluorescence-based CyQUANT Cell Proliferation Assay in 96-well plates. Significant cytotoxicity against HPDL fibroblast growth by Epiphany was both time- and concentration-dependent. On day 1, 800 microg/mL, the highest concentration of Epiphany, showed significant cytotoxicity (P < or = .001). By day 7, all concentrations greater than 25 microg/mL showed significant (P < or = .05) loss of viability. This study demonstrated increased Epiphany cytotoxicity with an increase in concentration or exposure time.     

平阳霉素固体植入式缓释剂间质化疗口腔鳞癌的实验研究

目的:制备含平阳霉素(PYM)原料药的可吸收固体植入式缓释剂,导入荷瘤裸鼠瘤体,实施组织内间质化疗,评价其抗肿瘤效应。方法:20只人舌鳞癌荷瘤裸鼠,随机分为5组,进行不同处理:①PYM固体缓释剂间质化疗;②PYM静脉化疗;③PYM瘤内注射;④缓释剂辅料瘤内植入;⑤生理盐水静脉注射空白对照。分别以肿瘤体积、组织学、分子生物学检测(TUNEL)和流式细胞术评价各组的抑瘤率、肿瘤细胞生长状态、凋亡指数及凋亡率,用SAS6.2软件包对抑瘤率、肿瘤细胞凋亡指数及凋亡率进行F检验。结果:①各组抑瘤率分别为(93.12±8.26)%(间质化疗组)、(57.57±2.90)%(静脉化疗组)、(55.20±4.28)%(瘤内注射组)和(22.48±3.74)%(辅料植入组);各组肿瘤细胞凋亡指数:15.88±0.80(间质化疗组)、6.80±0.59(静脉化疗组)、7.13±0.19(瘤内注射组)、3.0±0.37(辅料植入组)和2.2±0.18(生理盐水静注空白对照组);各组肿瘤细胞的凋亡率为:间质化疗组(27.80±13.78)%、瘤内注射组(13.13±0.35)%、静脉化疗组(6.81±0.89)%、辅料植入组(2.72±1.55)%和生理盐水静脉注射空白对照组(1.05±1.19)%。间质化疗组抑瘤率、肿瘤细胞凋亡指数及凋亡率显著高于其他各组(P<0.05);②间质化疗组肿瘤细胞增殖力下降,核分裂像罕见,细胞大量死亡或凋亡。结论:PYM固体植入式缓释剂间质化疗的抗癌效应优于其他给药方式,在体表肿瘤特别是口腔癌治疗中的应用前景乐观。     

Survivin基因在三氧化二砷诱导腺样囊性癌-M细胞凋亡中的作用

目的：体外观察三氧化二砷（As2O3）诱导人类腺样囊性癌高转移株（ACC-M）细胞凋亡过程中的作用,同时检测此过程中ACC-M内凋亡抑制基因Survivin表达水平的变化。方法：MTT法检测不同浓度As2O3作用不同时间对ACC-M细胞的抑制效应;流式细胞术观察As2O3诱导各组ACC-M细胞的凋亡率。RT-PCR检测Survivin基因mRNA的表达变化;Western blot检测判定Survivin蛋白水平的表达差异。结果：As2O3可明显抑制ACC-M细胞的生长,其抑制率呈浓度和时间依赖关系,细胞凋亡率也呈相同的趋势。RT-PCR和Western blot检测显示As2O3作用下,ACC-M内Survivin mRNA和蛋白表达明显受抑制,抑制程度也具有时间和剂量依赖性。结论：As2O3可通过促进细胞凋亡的方式使体外培养的ACC-M细胞生长受抑制,而受抑制的ACC-M内Survivin mRNA和蛋白表达均下降,推测Survivin基因在As2O3诱导的ACC-M细胞凋过程中起着重要作用。     

流式细胞仪法评价锶磷灰石细胞毒性

目的 尝试用流式细胞仪法探讨锶磷灰石的细胞毒性。方法 用流式细胞仪测定锶磷灰石浸提液作用细胞后细胞群落的DNA增殖指数。结果 增殖指数随着材料掺锶量的增加而增大,其中含锶100%的纯锶磷灰有利于产殖指数最大、1%锶磷灰石的增殖指数最小。结论 随掺锶量增加后锶磷灰石的细胞毒性也略增大。     

X-性染色体连锁凋亡抑制蛋白与口腔鳞癌细胞Tca8113化疗耐药的关系

目的研究X-性染色体连锁凋亡抑制蛋白（XIAP）在口腔鳞癌细胞Tca8113中的表达水平,并探讨XIAP表达与Tca8113细胞对化疗药物耐药性之间的关系。方法用平阳霉素（PYM）间歇性加药,逐步递增剂量,采用甲基噻唑基四唑（MTT）法检测药物处理前后细胞对PYM的敏感性,逆转录聚合酶链反应（RT-PCR）分别检测XIAP在药物处理前后的各Tca8113细胞组中的表达变化,并探讨XIAP与细胞耐药性的关系。结果Tca8113细胞在PYM间断作用下产生耐受,Tca8113-1-10组、Tca8113-10-10组耐药细胞对PYM的半数有效浓度（IC50）分别为（12.758±0.030）、（18.986±0.150）μg·mL-1。Tca8113-1-20、Tca8113-10-20组耐药细胞对PYM的IC50分别为（26.302±0.072）、（35.294±0.115）μg·mL-1。XAIP的表达水平与细胞对PYM的耐药有相关性（P<0.01）。结论XIAP在口腔鳞癌中的表达水平升高,可能与鳞癌的化疗耐药性有关,这可作为口腔鳞癌基因治疗的一个新靶点。     

Dual effect of nitric oxide in immortalized and malignant human oral keratinocytes: induction of apoptosis and differentiation

BACKGROUND: Nitric oxide (NO) is known to act cytostatically on several tumor cell when functioning as an effector molecule of activated macrophages, but the differential effects of NO on immortalized and malignant oral keratinocytes have not been examined. METHODS: We investigated the influence of NO on the proliferation, cell cycle, apoptosis, and differentiation of immortalized human oral keratinocytes (IHOK) and primary oral cancer cells (HN4) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, sulforhodamine B (SRB) assay, flow cytometry, nuclear DNA staining, and Western blotting. RESULTS: The MTT and SRB assays indicated inhibited growth of IHOK and HN4 cells that were treated with sodium nitroprusside (SNP) at concentrations higher than 1 mM but not at lower SNP concentrations. The higher concentrations of SNP up-regulated the apoptosis-related protein expression, which is consistent with the analyses of sub-G(1) phase arrest, annexin V-FITC (fluorescein isothiocynate) staining, nuclear staining, and DNA fragmentation. On the other hand, the lower concentrations of SNP enhanced the expression of keratinocyte differentiation markers in IHOK and HN4 cells. CONCLUSIONS: These data suggest that high concentrations of NO can inhibit the growth of IHOK and HN4 cells through the induction of apoptosis, while low concentrations of NO can induce cytodifferentiation. The dual effects of NO, namely, the induction of apoptosis or cytodifferentiation, have important implications for the possible anti-oral cancer treatment.     

重组人乳铁蛋白抑制口腔癌KB细胞增殖及诱导细胞凋亡

目的 研究重组人乳铁蛋白(recombinate human lactoferrin,rhLF)对人口腔KB细胞增殖及凋亡的影响,为口腔癌患者提供新的有效治疗途径提供参考。方法 采用CCK-8法检测不同浓度(0、12.5、25、50、100、200 μg/mL)的rhLF分别处理KB细胞24、48、72 h后对细胞增殖的影响;Immunofluorescence及Western blotting方法检测rhLF处理前后DNA损伤相关蛋白γ-H2AX的表达。并分别用0、50、200 μg/mL的rhLF处理KB细胞,14 d后观察其对集落克隆形成的影响;0、200 μg/mL rhLF处理KB细胞48 h后,收集细胞并使用线粒体膜电位检测试剂盒(JC-1)检测rhLF处理前后线粒体膜电位的变化。Annexin V-PI染色检测rhLF对口腔癌KB细胞凋亡的影响;Western blotting检测0、50、200 μg/mL的rhLF处理口腔癌KB细胞48 h后Parp、Caspase-3的表达情况。结果 rhLF处理KB细胞的增殖有显著的抑制作用。Immunofluorescence结果显示rhLF处理后γ-H2AX的表达明显增加,Western blotting结果也显示rhLF处理后γ-H2AX的表达成浓度依赖性增加。rhLF抑制KB细胞集落克隆的形成;JC-1荧光检测结果表明红绿荧光的相对比例降低,这种红绿荧光的相对比例降低提示膜电位下降;0、50、200 μg/mL的rhLF处理口腔癌KB细胞48 h的凋亡率为2.02%、7.60%、48.07%;同时rhLF刺激口腔癌KB细胞能诱导Parp、Caspase-3的激活。结论 rhLF能显著抑制KB细胞的增殖并且诱导其细胞凋亡,其机制与线粒体凋亡途径的激活应激有关。