5-氮胞苷对HSG细胞增殖抑制作用的体外研究

目的体外研究5-氮胞苷(5-azacytidine,5-azaC)对起源于人颌下腺闰管的HSG(HumanSalivaryGland,HSG,)细胞的增殖抑制作用,为5azaC治疗涎腺肿瘤奠定理论和实验基础。方法通过形态学研究、生长曲线的测定、细胞分裂指数测定、银染核仁组织者区分析、流式细胞仪、克隆形成率检测5azaC对HSG细胞的作用。结果光镜下培养的细胞为多边形,细胞可连接成片,胞膜清楚,核大,位于中间,核仁清楚,核仁1～2个。加入5azaC后,部分细胞脱落到培养液中。5μmol/L和10μmol/L5azaC对HSG细胞增殖抑制率分别为85%和95%,细胞分裂减慢,细胞软琼脂克隆形成率降低,流式细胞仪结果表明HSG细胞主要被抑制在G1期。结论5azaC在体外能抑制HSG细胞的增殖。     

5-azaC对HSG细胞的诱导分化作用

目的 体外研究5-氮胞苷（5-azacytidine，5-asaC）对起源于人颌下腺闰管的HSG（human salivary gland，HSG）细胞的诱导分化作用，为应用5azaC对涎腺肿瘤进行分化治疗奠定理论和实验基础。方法 用不同浓度的5azaC处理HSG细胞，通过形态学观察和RT-PCR检测HSG细胞的分化程度。结果 加入5azaC后，细胞胞浆内出现颗粒状物质，而对照组则没有出现，RT-PCR产物电泳结果可见经过诱导的HSG细胞在mRNA水平表达α-唾液淀粉酶，未诱导的HSG细胞不表达α-唾液淀粉酶。结论 5azaC在体外能诱导HSG细胞分化。     

选择性环氧化酶-2抑制剂抑制人舌癌细胞增殖和浸润的体外实验研究

目的探讨选择性COX-2抑制剂Ethodolac对人舌癌细胞SAS的增殖和浸润能力的抑制作用。方法将人低分化舌鳞状上皮细胞癌细胞株(SAS)与不同浓度的选择性COX-2抑制剂Ethodolac共同培养,染色后测定595nm下的吸光度。比较不同浓度Ethodolac下的细胞增殖情况。同时,利用Boyden小室测定Ethodolac对SAS细胞浸润能力的影响。结果Ethodolac可以明显降低SAS细胞的增殖和浸润能力(P<0.05),作用效果呈现时间剂量依赖关系。结论本研究为COX-2在肿瘤发生、发展中的作用提供了一定依据,而且也通过COX-2选择性抑制剂Ethodolac对人舌癌细胞SAS增殖和浸润的抑制,提示选择性COX-2抑制剂在口腔癌的预防和治疗中的作用前景。     

FAP-1在5-azaC诱导的HSG细胞凋亡中的作用

目的体外研究FAP-1在5-氮胞苷（5-azacytidine,5-azaC）诱导的HSG（human salivary gland。HSG,人涎腺导管细胞,起源于人颌下腺闫管细胞）细胞凋亡中的作用,为应用5-azaC对涎腺肿瘤进行治疗奠定理论和实验基础。方法HSG细胞转染FAP-1反义寡核苷酸前后分别用5-azaC处理,观察转染前后FAP-1表达的变化,同时观察5-azaC对转染前后细胞凋亡作用是否发生变化。结果在形态上转染后的细胞与转染前无明显区别,转染FAP-1反义寡核苷酸链的HSG细胞FAP-1表达较转染前减弱,转染后HSG细胞对5-azaC比转染前敏感,加入5umol／L5-azaC后,凋亡细胞数增加。结论FAP-1在5-azaC诱导的HSG细胞凋亡中起一定作用。     

苦参碱对人涎腺腺样囊性癌ACC-M细胞增殖抑制的研究

目的探讨中药有效成分苦参碱(Mat)对人涎腺腺样囊性癌(SACC)细胞系ACC-M细胞增殖的抑制作用。方法体外培养ACC-M细胞,用不同质量浓度Mat溶液对体外培养的ACC-M细胞进行处理。通过MTT比色法和流式细胞仪细胞周期检测来测定不同质量浓度Mat溶液对ACC-M细胞增殖的抑制作用。结果经Mat处理后,ACC-M细胞生长明显受到抑制,细胞周期阻滞于G0/G1。结论Mat对ACC-M细胞的增殖有显著的抑制作用,其抑制作用的发生可能与其细胞周期阻滞有关。     

EGFRmAb抑制口腔鳞癌细胞增殖的研究

目的 研究表皮生长因子受体单克隆抗体(EGFRmAb)对口腔鳞癌细胞增殖的抑制作用及其作用机理。方法 对Tca8113舌鳞癌细胞系给予不同浓度的EGFRmAb处理，通过分析细胞生长曲线和克隆形成抑制率，观察EGFRmAb225对舌鳞癌细胞的增殖抑制作用，并通过分析细胞周期再分配及细胞周期相关蛋白p27kipl的表达，探讨其作用机制。结果 EGFRmAb可抑制舌鳞癌细胞的增殖和克隆形成，可以导致G1期细胞比例上升和S期细胞比例下降，并使p27kipl的表达上调。结论 EGFRmAb可以抑制舌鳞癌细胞的增殖，其机制与细胞的G1期阻滞有关。     

TNF-α对HSG细胞作用的体外研究

目的体外研究TNF-α对导管起源的HSG（human salivary bland,HSG）细胞的增殖抑制作用,为TNF-α治疗涎腺肿瘤奠定理论和实验基础。方法通过形态学研究、生长曲线的测定、细胞分裂指数测定、嗜银蛋白染色分析、流式细胞仪、克隆形成率等观察TNF-α对HSG细胞的作用。结果光镜下培养的正常HSG细胞为多边形,细胞可连接成片。加入TNF-α后,部分细胞发生形变,脱落到培养液中。1ngl/L、10ngl/L和50ng/L TNF-α对HSG细胞增殖抑制率分别为5%、72%和81%;细胞分裂指数测定、嗜银蛋白染色分析、克隆形成率等结果表明细胞生长受到抑制;流式细胞仪结果表明HSG细胞主要被抑制在G1期。结论 TNF-α在体外能抑制HSG细胞的增殖。     

SFN联合5-Fu对ACC—M细胞增殖抑制的实验研究

目的：评价莱菔硫烷（SFN）与5-氟尿嘧啶（5-Fu）对人高转移唾液腺腺样囊性癌细胞（ACC—M）生长的抑制作用，同时检测参与诱导抗癌药物耐药性的核因子KB（NF—kB）的表达。方法：中位效应法评价SFN与5-Fu联用对ACC—M细胞增殖的影响．NF—kBp65表达采用Western印迹法检测。采用SPSS10．0软件包对所得数据进行t检验和方差分析。结果：SFN与5-Fu单独作用于ACC—M时的中位抑制浓度分别为（38．58±4．97）umol／L和（120．90±12．96）umol／L。两者联合使用时，对ACC—M细胞生长呈现协同抑制作用，并且引起细胞核NF—kBp65表达显著降低。与对照组相比．两药联合使用时．NF—kBp65的表达约降低5倍，P〈0．05。结论：SFN与5-Fu联合使用可协同抑制ACC—M细胞增殖．该作用与SFN、5-Fu协同抑制细胞核NF—kBp65的表达有关。     

过表达大肿瘤抑制因子2对口腔鳞状细胞癌细胞增殖和凋亡的影响

目的 探讨过表达大肿瘤抑制因子2（LATS2）基因对口腔鳞状细胞癌（OSCC）细胞增殖和凋亡的影响。方法 应用慢病毒介导的基因过表达技术,使用LATS2过表达慢病毒感染SCC-25细胞株。通过CCK-8检测过表达LATS2对细胞增殖的影响;流式细胞术检测LATS2基因过表达后细胞凋亡情况;Western blot检测LATS2过表达后对凋亡相关蛋白Bax和Bcl-2表达的变化。结果 转染pEGFP-LATS2细胞的LATS2基因表达明显上调;pEGFP-LATS2组细胞增殖明显受到抑制;流式细胞术检测pEGFP-LATS2组凋亡率明显高于空白对照组（control）和空载体组（pEGFP-control）（P<0.05）。与control和pEGFP-control组相比,pEGFP-LATS2组Bax蛋白上调,而Bcl-2蛋白下调。结论 过表达LATS2基因可明显抑制SCC-25细胞增殖并促进细胞凋亡。     

尼古丁抑制成牙本质细胞增殖及作用机制的研究

目的观察尼古丁对成牙本质细胞增殖的抑制作用,并检测其对成牙本质细胞中Ca2+浓度的影响,以探讨尼古丁抑制成牙本质细胞的分子机制。方法体外培养成牙本质细胞系MDPC- 23细胞, 按每毫升2×104个接种,随机分为实验组和对照组,对照组不加任何刺激,实验组施加质量浓度为100 μg/mL尼古丁,并于8 h后加入浓度为10 μmol/L BrdU进行细胞周期标记,刺激24 h后固定细胞,行免疫荧光抗BrdU染色,碘化丙锭（PI）复染胞核,荧光显微镜下计数细胞总数与BrdU阳性细胞数,计算S期阳性细胞率并进行统计学分析。培养成牙本质细胞于特制培养皿中,施加质量浓度为100 μg/mL尼古丁刺激,激光共聚焦显微镜下检测成牙本质细胞中Ca2+浓度的动态变化。结果实验组、对照组S期阳性细胞率分别为36.3%、48.2%,实验组显著低于对照组。尼古丁刺激后,成牙本质细胞中Ca2+浓度迅速升高,在较高水平维持一段时间后缓慢下降。结论尼古丁可抑制成牙本质细胞增殖,这种作用同尼古丁升高成牙本质细胞中Ca2+浓度有关。     

5-氮胞苷对HSG细胞的诱导凋亡作用

目的体外研究5-氮胞苷（5-azacytidine,5-azaC）对起源于人颌下腺闰管的HSG（human salivary gland,HSG）细胞的诱导凋亡作用,为应用5azaC对涎腺肿瘤进行治疗奠定理论和实验基础。方法用不同浓度的5-azaC处理HSG细胞,观察5-azaC在非毒性浓度下,能否诱导HSG细胞发生凋亡。结果加入5-azaC后48h,光镜下可见到少数细胞体积缩小,胞浆向外突起,类似出芽及发泡状;琼脂糖凝胶电泳结果显示经5-azaC作用72h的基因组DNA呈现出＂梯状＂图像,基因组DNA被内切酶切成了180bp～200bp及其整倍数的片断;流式细胞仪检测结果显示细胞在G1期前面出现亚二倍体峰;透射电镜结果显示5-azaC处理组细胞可见核内凋亡小体;免疫组化结果显示5-azaC可以抑制P53、Bcl-2和FAP-1的表达;Western blot结果显示5-azaC处理后可使FAP-1的表达降低。结论5-azaC在体外能诱导HSG细胞凋亡。     

Caffeine increases the expression of cystatin SN in human submandibular acinar-like HSG cells

ObjectiveThe study aimed at evaluating in vitro the effect of caffeine on expression of cystatin SN, a potential marker of sensitivity to bitterness in humans.MethodsDifferentiation of human submandibular gland (HSG) cells was induced by culturing cells on Matrigel. Caffeine cytotoxicity was assessed over 3 days by the Resazurin test. Finally, effects of 5, 50 and 100 μM caffeine exposure on cystatin SN expression were explored over 3 days by ELISA.ResultsAt concentrations relevant to human adult plasma levels (5, 50 and 100 μM), caffeine did not affect cell viability whether cells were differentiated or not. Cystatin SN levels were overall higher in differentiated cells and increased with time in both conditions. There was a significant (p < 0.001) effect of caffeine on cystatin SN expression specifically in differentiated cells.ConclusionsThe HSG cell line proved to be a relevant tool to study in vitro the effect of caffeine at concentrations consistent with dietary intake in human subjects. The results suggest that salivary cystatin SN abundance may depend on caffeine intake, with possible consequences on taste sensitivity.     

Alkalization produced by high-dose carbachol in HSG cell line is independent of Ca2+

OBJECTIVES: The aim of this investigation was to clarify the mechanism of alkalization induced by carbachol in HSG cells. MATERIALS AND METHODS: Cells of the HSG cell line derived from a human submandibular gland adenocarcinoma and those of the A-431 human epidermoid carcinoma cell line were loaded with a fluorescent pH indicator, BCECF/AM, and the change in the intracellular pH of adherent cells and suspended ones were measured following stimulation with various concentrations (10(-7) M to 10(-2) M) of neurotransmitters (carbachol, noradrenaline, and isoproterenol). RESULTS: Isoproterenol did not cause alkalization of either cell type, whereas, noradrenaline and carbachol alkalized both types over the concentration ranges of 10(-6) M to 3 x 10(-3) M (HSG cell by noradrenaline), 10(-7) M to 2 x 10(-4) M (A-431 cell by noradrenaline), and 7 x 10(-5) M to 10(-4) M (A-431 cell by carbachol). On the other hand, alkalization induced by carbachol in the HSG cells was recognized at concentrations higher than 6 x 10(-5) M, and it showed no upper limit in terms of carbachol concentration. This high-dose carbachol alkalization was not eliminated by preincubation with nifedipine (100 microM), a Ca2+ channel blocker, or with thapsigargin (100 microM), a microsomal Ca(2+)-ATPase inhibitor. CONCLUSIONS: The alkalization system induced by carbachol in the HSG cell was quite different from that in the A-431 cell, and that induced by high-dose carbachol in HSG cells appeared to be independent of intracellular Ca2+. These findings will be useful to clarify the mechanism of salivary secretion stimulated by neurotransmitters.     

5-FU聚乳酸纳米微球对人舌癌Tca83细胞增殖及凋亡的影响

目的:探讨5-FU聚乳酸纳米微球抑制人舌癌Tca83细胞增殖及诱导其凋亡的作用。方法:5-FU聚乳酸纳米微球、5-FU及空白对照组分别作用于Tca83细胞,四唑盐(MTT)比色法检测细胞存活率;吖啶橙(AO)荧光染色法检测细胞凋亡情况,并计算凋亡指数(AI);流式细胞仪(FCM)检测细胞周期的变化。结果:5-FU与5-FU聚乳酸纳米微球组自第3天起与对照组之间存在显著性差异(P<0.05),且差异随作用时间的延长、作用浓度的增加而增大,其中5-FU组持续到第7天其后差异的变化不再具有统计学意义;而5-FU聚乳酸纳米微球组则一直持续到第11天且差异的增加始终具有统计学意义,同时自第7天起与同条件下5-FU各浓度组比较差异亦有显著性(P<0.01)。结论:5-FU聚乳酸纳米微球可抑制Tca83细胞增殖并诱导其凋亡,其作用呈剂量和时间依赖性,且作用时间较5-FU延长、作用效果较5-FU提高。     

DNA damage and apoptosis induced by Pteridium aquilinum aqueous extract in the oral cell lines HSG and OSCC-3

Background:  Bracken fern ( Pteridium aquilinum ) has been consumed by humans and animals for centuries. However, its consumption is associated with a high incidence of cancer in the upper digestory tract of different species. Although the oral cavity is the first site of contact with ingested toxic substances, the interaction of bracken fern composites with oral cell lines has not yet been studied.
Methods:  In order to study the biological responses of oral cells exposed to bracken fern, we evaluated the genotoxic and cytotoxic effects of a bracken fern aqueous extract in oral cell lines. Human submandibular gland (HSG) and human oral epithelium cells (OSCC-3) cells were treated with three different concentrations of the extract. DNA damage was determined by the comet assay, and cellular morphology was examined by light microscopy. Apoptotic changes were evaluated by transmission electron microscopy and TUNEL assay.
Results:  The comet assay revealed that the extract was genotoxic for both cell lines but the results were not dose-dependent. The morphological and ultrastructural analyses showed that the extract caused conspicuous alterations in both cell types: uncommon chromatin condensation, nuclear picnosis, cellular volume decrease, nuclear envelope disruption, formation of numerous vacuoles of different sizes and apoptotic bodies. The TUNEL assay confirmed apoptosis induction.
Conclusions:  These results demonstrate that the extract was cytotoxic to HSG and OSCC-3 cells, and that cellular degeneration occurred mainly by apoptosis. We believe that oral cells could trigger apoptosis after bracken fern induced DNA damage, in order to avoid the malignant transformation.     

Mechanism of Fas-mediated cell death and its enhancement by TNF-alpha in human salivary gland adenocarcinoma cell line HSG

Fas-mediated cell death in a human salivary gland adenocarcinoma cell line (HSG) was induced by treatment of the cells with agonistic anti-Fas antibody (CH-11), and this cell death was enhanced by pretreatment with tumor necrosis factor alpha (TNF-alpha). The mode of cell death was apoptosis, because it was accompanied by caspase activation and the cleavage of poly(ADP-ribose) polymerase. The TNF-alpha treatment of the cells increased the expression of Fas, which was accompanied by the activation of nuclear factor kappaB (NFkappaB). These results suggest that the enhancement of the apoptosis caused by TNF-alpha resulted from increased sensitivity of the HSG cells to CH-11-mediated apoptosis due to induction of Fas protein by TNF-alpha via the activation of NFkappaB. In order to elucidate the apoptosis signaling pathway, we examined the effect of various caspase inhibitors on the apoptosis induced by CH-11. Fas-mediated apoptosis of HSG cells was slightly inhibited by the caspase-9 inhibitor although it was mainly inhibited by that for caspase-8. Based on this finding, we consider CH-11-induced apoptosis in HSG cells to be mainly mediated by the type I death signaling pathway that is caused by a caspase cascade initiated by the activation of caspase-8 at the death-inducing signaling complex (DISC).