As2O3联合FP99诱导骨肉瘤细胞MG-63凋亡的初步研究

采用噻唑蓝(MTT)法发现FP99能够协同三氧化二砷(As2O3)抑制人骨肉瘤细胞MG-63的生长,并采用流式细胞计数进一步证实FP99加强As2O3对MG-63细胞凋亡的诱导作用;进而检测了Caspase-3的活性,发现As2O3联合FP99作用MG-63细胞后,Caspase-3的活性显著提高,提示我们As2O3联合FP99增加MG-63细胞的凋亡率的机制可能是通过提高Caspase-3激酶活性,从而促进了整个凋亡通路的激活,最终导致细胞凋亡的增加。     

Effect of thrombin on the apoptosis of hippocampal neurons in vitro

Hippocampal neurons were treated by thrombin and thrombin receptor activating peptides (TRAP). Cell survival rate was decreased in a dose-dependent manner by MTT assay. The numbers of apoptotic cell and apoptotic rate of hippocampal neurons treated by different concentrations of thrombin were increased in a dose-dependent manner by terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end-labeling (TUNEL) method and Flow Cytometry. When the concentration of thrombin is 40 U/mL, TUNEL positive cells and apoptotic rate of hippocampal neurons reached peak value, were 27.3±4.0 and (29.333±4.633)%, respectively. Immunocytochemistry assay show that Bcl-2 protein expression was down-regulated and Bax protein expression was up-regulated with the concentration of thrombin increased. TRAP can mimic the effect of thrombin to induce apoptosis on hippocampal neurons. These data demonstrated that thrombin induced hippocampal neuron apoptosis in a dose-dependent manner through activating protease-activated protein-1 (PAR-1). The change in expression of Bcl-2 and Bax was related with the effect of high concentration thrombin induced apoptosis on hippocampal neurons. Foundation item: Supported by the Natural Science Foundation of Hainan Province (N30215) Biography: YANG Wen-qiong (1968-), female, Ph.D. candidate, research direction: cerebrovascular disease.     

HMBA对人成骨肉瘤MG-63细胞形态结构和终末分化指标表达的影响

应用环六亚甲基双乙酰胺处理人成骨肉瘤MG-63细胞,观察MG-63细胞处理前后形态与超微结构及其相关终末分化指标的表达变化.实验结果显示,经5 mmol/L HMBA处理后,MG-63细胞体积增大,趋于扁平铺展状态,细胞大小较为一致,排列较为规则,细胞核形态规则,核质比例减小,核仁减少,核内异染色质减少,常染色质增多,细胞核内的线粒体和高尔基体较为发达,内质网数量增多,细胞表面的微绒毛减少,在成熟细胞中可见钙化糖原颗粒,细胞表面出现钙化小泡沉积,并且形成典型的骨结节.常规细胞化学和免疫细胞化学检测显示,HMBA处理后的细胞中Ⅰ型胶原纤维、骨钙素、骨粘素的表达显著增加,实验结果表明HMBA能够有效诱导人成骨肉瘤细胞的分化,并促进其终末分化指标的表达.     

细胞悬液注射法与组织块移植法建立骨肉瘤动物模型的比较

 为建立裸鼠胫骨原位骨肉瘤模型,并比较分析MG-63细胞悬液局部注射以及肿瘤组织块移植两种方法的成瘤特性,在注射组体内连续传代筛选MG-63细胞系获得高成瘤率的细胞,将MG-63细胞悬液(25μL,约1×10⁵个)注射于裸鼠胫骨上段髓腔内;组织块移植组将MG-63细胞皮下成瘤瘤组织块接种于裸鼠胫骨上段髓腔内。随后观察两种方法建立的胫骨骨肉瘤原位模型不同的成瘤特性,并采用影像学等手段比较两种方法的成瘤率和成瘤特点。结果表明,术后3周左右可见局部肿瘤形成,4周后通过小动物X线技术可见胫骨中上段溶骨与肿瘤样骨形成,同时在光学显微镜下可见典型的骨肉瘤病理特征。细胞悬液法成瘤率85%,组织块法成瘤率95%,两种方法成瘤率的差异有统计学意义(P<0.05),模型组血清碱性磷酸酶(ALP)明显高于对照组(P<0.01)。两种方法均可形成裸鼠胫骨原位骨肉瘤模型,采用组织块移植法成瘤率较高,瘤体生长速度较快,而且对骨皮质及周围软组织的破坏力较强,转移率高。     

Recombinant sTRAIL induces cell death of tumor cell lines as well as primary cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a new member of TNF family. It was reported that TRAIL could induce apoptosis of tumor cells but not normal cells in tissue culture system. To further study the biological activity and potential clinical significance, a recombinant soluble TRAIL (rsTRAIL) has been expressed stably in E. coli after transformation of pET28b vector containing the extracellular domain of TRAIL. The yield of rsTRAIL is approximately as high as 60% of whole bacterial proteins. The rsTRAIL, purified by Ni+ -agarose affinity chromatography, could remarkably trigger apoptosis at the concentrations of 0.1-1 μg/mL in all 7 tumor cell lines tested in vitro. However, this killing activity has not been observed in mouse fibroblast cell line (NIH3T3) as normal control. Further investigation shows that the rsTRAIL could also kill primary tumor cells isolated freshly from patients with cardiac cancer, breast cancer and malignant thymoma, while the normal human peripheral blood lymphocytes are not killed under the same conditions. These results provide new evidence that rsTRAIL could induce apoptosis of tumor cells specifically and it could be a new promising medicine for tumor therapy.     

HMBA对人成骨肉瘤MG-63细胞增殖和相关基因表达的影响

研究了环六亚甲基双乙酰胺对人成骨肉瘤MG-63细胞的增殖和相关基因表达的影响.实验结果表明HMBA可明显抑制MG-63细胞的增殖,细胞生长抑制率达50.69%,分裂指数抑制率达58.8%,增殖细胞核抗原的表达降低,细胞周期被阻滞在G0/G1期.免疫细胞化学染色结果显示,经HMBA处理之后,与增殖分化调控有关的癌基因c-myc、c-fos、c-erbB-2、mtp53的表达降低、抑癌基因p21WAF1/CIP1、p16、rb的表达升高.研究结果表明,HMBA能够有效抑制人成骨肉瘤MG-63细胞的增殖活动,其对细胞增殖的抑制作用与HMBA下调c-myc、c-fos、c-erbB-2、mtp53等癌基因以及上调p21WAF1/CIP1、p16、rb等抑癌基因的表达,从而调控细胞周期有重要关系.     

Involvement of Oxidative Stress and Down-Regulation of Bcl-2 in Arachidonic Acid-Induced Apoptosis in HUVECs

0IntroductionAraacicdhi,diosn fiocu ancidd p(reAdAo)m i,naanntelsys eantt itahle p sonl-y2u npsoastiutiroante doff actetl-ylular phospholipids . Normal free AAconcentrationin humanblood ranges from5 .8μmol/Lto 49 .3μmol/L[1].It is re-leased mostlythroughactivation of phospholipase A2by physi-ological and pathological sti muli[2]. Free AAcan be metabo-lizedinto various eicosanoids via specific enzymes such as cy-clooxygenases ( COX) , lipoxygenase and cytochromesP-450[3]. During AA met…     

可溶性人TRAIL蛋白诱导肝癌细胞的凋亡

为研究可溶性人TRAIL蛋白（sTRAIL）对肝癌细胞株SMMC7721的生长抑制效应及凋亡诱导作用．采用显微镜、台盼蓝排斥试验、MTT比色试验、TUNEL法和DNA断裂实验等方法检测细胞增殖和细胞凋亡．通过显微镜观察到核染色质凝集等凋亡的形态学变化,台盼蓝排斥试验、MTT比色试验结果显示,sTRAIL蛋白可显著抑制SMMC7721细胞的生长和增殖,并且TUNEL法检测到经sTRAIL处理后的细胞凋亡指数与对照比较有显著差异,DNA断裂实验亦观察到典型的DNA梯形条带,这些结果提示sTRAIL可诱导肝癌细胞株SMMC7721发生凋亡,具有抗肝癌的作用．     

文殊兰弱碱性生物碱的抗肿瘤活性研究

研究文殊兰中弱碱性生物碱对不同肿瘤细胞的抑制作用,并筛选出敏感细胞株,观察凋亡形态;MCF-7细胞株、SMMC7721细胞株、SGC7901细胞株、HepG-2细胞株,MTT溶液,DMSO,Hoechst33258等;利用MTT法检测文殊兰弱碱性生物碱对上述肿瘤细胞的抑制作用以及Hoechst 33258染色法观察细胞凋亡的形态学变化;文殊兰弱碱性生物碱对上述细胞株均有明显的抑制作用,对HepG-2细胞的抑制作用更为明显,给药72 h后测定IC50=9.374 323μg/mL;Hoechst 33258染色法观察细胞出现凋亡小体,有凋亡现象发生.文殊兰弱碱性生物碱MCF-7细胞株、SMMC7721细胞株、SGC7901细胞株、HepG-2细胞株具有不同程度的抑制作用,且呈现一定的剂量依赖性,对HepG-2细胞的抑制作用最强,表现出良好的抗肿瘤活性.     

2-ME抑制人子宫内膜癌细胞株增殖的研究

目的　探讨2-甲氧雌二醇(2-ME)对人子宫内膜癌细胞株KLE细胞体外增殖和凋亡的抑制作用.方法选用人子宫内膜癌细胞株KLE进行体外培养,实验组加入不同浓度2-ME的培养液,对照组不含2-ME.用四甲基偶氮唑蓝(MTT)比色法观察2-ME对人子宫内膜癌细胞株KLE增殖的抑制作用;药物作用后的克隆形成实验;电子显微镜(电镜)观察细胞形态变化;流式细胞仪(FCM)观察细胞的凋亡率及细胞周期的变化.结果2-ME浓度为10.0～50.0μM时,明显抑制KLE细胞的增殖(P<0.01),并具有时间依赖性和剂量依赖性.2-ME作用后G0/G1期细胞增加,并伴随G0/G1期细胞的增加,出现细胞凋亡峰和凋亡率的升高(P<0.05).电镜下观察到KLE细胞染色体边集、核固缩、凋亡小体.结论2-ME对人子宫内膜癌KLE细胞株增殖有抑制作用,并能促进其凋亡.     

Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast

The use of plants as bioreactors for the expression oftherapeutic proteins has developed into a new field in biotechnology research[1]. Plant systems provide simple genetic manipulation, low production costs, and low risk of contamination by animal viruse…     

OA对人肝细胞HL-7702和肝癌细胞Bel-7402增殖的影响和对细胞凋亡的诱导作用研究

 以人肝细胞HL-7702和肝癌细胞Bel-7402为研究对象,通过MTT法、细胞外部形态观察、Annexin V.FITC&;PI双染法及TUNEL法等方法,研究了腹泻性贝毒的主要组分大田软海绵酸（Okadaic acid, OA）对其增殖的影响及对细胞凋亡的诱导作用。结果表明：OA对人两株细胞的增殖均有显著的抑制作用,24 h IC₅₀分别为65 ng/mL和60 ng/mL,且这种抑制作用呈剂量依赖性。OA导致两株细胞的形态均发生明显变化：细胞变圆,脱壁,细胞膜形成泡状突起,最终形成膜包裹的凋亡小体。Annexin V.FITC和PI双染法及TUNEL法检测均发现：OA能够诱导两株细胞发生不同程度的凋亡,且凋亡细胞的比例与OA的浓度相关。以上结果表明：OA能够通过抑制细胞增殖和诱导细胞凋亡而对人肝细胞和肝癌细胞造成影响,且这种影响的程度与OA的浓度和作用时间密切相关。     

利多卡因对人角膜上皮细胞的毒性作用及其机理研究

利用不同浓度利多卡因（ｌｉｄｏｃａｉｎｅ）处理体外培养的人角膜上皮（ＨＣＥＰ）细胞系细胞,利用光镜观察、ＭＴＴ、荧光染色、ＤＮＡ电泳、ＴＵＮＥＬ、流式细胞仪和透射电镜方法研究了利多卡因对 ＨＣＥＰ细胞的毒性作用及其机理。光镜观察和 ＭＴＴ检测结果显示,质量浓度 １２５～１０００ｇ／Ｌ的利多卡因对 ＨＣＥＰ细胞具有显著的毒性作用,并具有浓度和时间依赖性;ＡＯ／ＥＢ荧光双染色结果显示,质量浓度 ０６２５～１００００ｇ／Ｌ的利多卡因可引起 ＨＣＥＰ细胞的质膜通透性显著提高,细胞凋亡率也具有浓度和时间依赖性;ＤＮＡ电泳和 ＴＵＮＥＬ检测结果显示,利多卡因能引起 ＨＣＥＰ细胞发生 ＤＮＡ断片化;ＴＥＭ观察结果显示,利多卡因能引起 ＨＣＥＰ细胞的超微结构出现了凋亡细胞的形态结构特征,如胞质空泡化、染色质浓缩、线粒体膨胀且嵴的结构紊乱、出现凋亡小体等;ＡｎｎｅｘｉｎＶ／ＰＩ染色的流式细胞仪检测结果显示,利多卡因能引起 ＨＣＥＰ细胞质膜中的磷脂酰丝氨酸（ＰＳ）发生外翻变化;ＥＬＩＳＡ检测结果显示,利多卡因还能引起 ＨＣＥＰ细胞中胱冬肽酶3、8、9、10表达量的增加,表明利多卡因确能引起 ＨＣＥＰ细胞发生细胞凋亡,而不是细胞坏死。由此可见,利多卡因在质量浓度大于 0.625ｇ／Ｌ时对 ＨＣＥＰ细胞具有显著的细胞毒性,并具有浓度和时间依赖性,且其毒性作用的发挥是通过诱导细胞凋亡实现的,在眼科临床应用中具有很大的毒副作用,应谨慎使用。     

The molecular mechanism of different sensitivity of breast cancer cell lines to TRAIL

Although Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of various cancer cells, some caner cell lines are resistant to TRAIL-induced cell death. To investigate the molecular mechanisms underlying TRAIL-resistance, two human breast cancer cell lines, MCF-7 (resistant to TRAIL) and MDA-MB-231 (sensitive to TRAIL), were used as a model system to analyze the different sensitivities to TRAIL cytotoxicity. PKCδ inhibitor rottlerin, but not MEK and ERK1/2 inhibitor U0126 nor PI3K inhibitor LY294002, was shown to enhance TRAIL-induced apoptosis in MCF-7 cells significantly, suggesting that PKCδ might play an important role in the resistance of MCF-7 cells to TRAIL. In contrast, rottlerin, U0126, and Ly294002 had no effect on MDA-MB-231 apoptosis induced by TRAIL under the same conditions. Further experiment showed that the combination of rottlerin and TRAIL cleaved PARP in the MCF-7 cells synergistically, but not in the MDA-MB-231 cells. The role of PKCδ in TRAIL-resistant MCF-7 cells was confirmed by knocking down the endogenous PKCδ expression using RNAi technology. Furthermore, caspase-3 reconstitution in MCF-7 cells was unable to alter PKCδ expression, suggesting that innate caspase-3 deficient in the cells does not cause PKCδ high expression. These data provide evidence for the first time that PKCδ plays a critical role in breast cancer cell lines to TRAIL cytotoxicity.     

Recombinant soluble TRAIL induces apoptosis of cancer cells

TRAIL is a tumor necrosis factor family member that selectively induces apoptosis of cancer cells but not of normal cells. To develop TRAIL into a potential cancer drug, three different sizes of soluble TRAIL fragments, including sTRAIL(74—281), sTRAIL(95—281) and sTRAIL(101—281), were expressed in E. coli and purified to homogeneity. Apoptosis assays indicated that sTRAIL(95—281) and sTRAIL(101—281), but not sTRAIL(74—281), can potently induce apoptosis of various cancer cell lines in 6 h, suggesting that the N-terminal fragment of aa101 has inhibitory effect on TRAIL-induced apoptosis. Moreover, we found that some cancer cells were resistant to TRAIL and the resistant cells could be converted into sensitive cells by treatment with the protein synthesis inhibitor cycloheximide, suggesting that one or more short-lived proteins are responsible for cells’ resistance to TRAIL.     

神经毒素MPP+诱发PC12细胞凋亡的实验研究

采用细胞体外培养、噻唑蓝(MTT)比色法、荧光染色技术、透射电镜、流式细胞技术结合的方法,研究了神经毒素MPP 对PC12细胞增殖和凋亡的影响,为进一步研究药物的神经保护作用提供实验模型.结果表明,MPP 对PC12细胞的诱导凋亡作用呈时间和剂量相关性.0.20mM的MPP 作用于PC12细胞48h后,细胞的存活率降低为60%,细胞核发生凝集和断裂,超微结构发生了一系列的变化,呈现出明显的凋亡特征.流式细胞技术的Annexin v-FTTC和PI双染法显示,活细胞占20.2%,晚期凋亡/坏死细胞占51%,早期凋亡细胞占6.75%.提示,MPP 能够诱导PC12细胞凋亡,是研究神经保护作用药物很好的体外实验模型.     

野西瓜总皂苷诱导SGC-7901凋亡的初步研究

探讨野西瓜总皂苷诱导人胃癌SGC-7901细胞的凋亡作用.采用MTT法检测野西瓜总皂苷对SGC-7901细胞增殖的抑制作用;采用荧光倒置显微镜观察细胞凋亡的形态学变化;采用流式细胞术分析野西瓜总皂苷对SGC-7901细胞凋亡率的影响;采用激光共聚焦显微镜观测细胞内[Ca2+]i的变化.5、25、50、100、200和400 μg/mL的野西瓜总皂苷作用于SGC-7901细胞72 h后,野西瓜总皂苷可抑制SGC-7901细胞的增殖,其IC50为31.542 μg/mL;野西瓜总皂苷作用SGC-7901细胞40 h后,细胞出现早期凋亡细胞的形态;15、30、60 μg/mL的野西瓜总皂苷作用于SGC-7901细胞72 h后,细胞凋亡率分别为58.6%、92.298%、95.898%;15、30、60 μg/mL野西瓜总皂苷作用SGC-7901细胞24 h后,细胞内[Ca2+]i升高,并随野西瓜总皂苷给药剂量的增加而升高.野西瓜总皂苷能够促进SGC-7901细胞的凋亡,其作用机制可能与野西瓜总皂苷升高细胞内[Ca2+]i有关.     

Cinobufacini-induced HeLa cell apoptosis enhanced by curcumin

When used in combination with certain chemotherapies, curcumin has been shown to increase apoptosis in several cancer cell lines. Here, we report the combined effects of curcumin and cinobufacini on human cervical carcinoma cells. The aim of this study was to examine whether curcumin could enhance apoptosis induced by cinobufacini. 3-(4,5-Dimethylthiazol-2-y1)-2,5- diphenytetrazolium bromide (MTT) assays revealed that the growth and proliferation of HeLa cells could be inhibited by 75% after a combined treatment of 25 μg/mL cinobufacini and 8 μg/mL curcumin. The combined treatment is 3 times more effective than treatment with 25 μg/mL cinobufacini alone. Annexin V-FITC/PI staining, morphological changes and immunofluorescence verified a significant enhancement in cinobufacini-induced apoptosis when cells were also exposed to curcumin. The data showed that the proportion of early apoptotic cells significantly increased from 15.43% in cells treated only with 25 μg/mL cinobufacini to 49.2% in cells treated with 25 μg/mL cinobufacini and 8 μg/mL curcumin. Moreover, compared with treatment of only 25 μg/mL cinobufacini, ROS production increased 1.7-fold, the intracellular free Ca 2+ concentration increased 1.5-fold, and the mitochon- drial membrane potential decreased by 20% in the combined treatment. Simultaneously, the atomic force microscopy (AFM) re- sults suggest that cells treated with a combination of cinobufacini and curcumin varied significantly in shape and ultrastructure. Collapsed cells with leaking cytoplasm, blebbing pores and emerging apoptotic bodies were prevalent. The nanoparticle size increased from 70 nm when the cells were treated with 25 μg/mL cinobufacini to 190 nm when the cells were treated with 25 μg/mL cinobufacini and 8 μg/mL curcumin. The size increase resulted in the cell membrane becoming considerably rough. These results can improve our understanding of combination treatments. Specifically, the combination of cinobufacini and curcumin may potentially find use as a novel cervical carcinoma treatment. Additionally, AFM is a powerful tool that can be used to explore cellular morphologies and ultrastructures.     

天然丝素制备三维多孔支架

研究盐沥滤法制备三维多孔丝素支架,在丝素多孔支架上培养人成骨肉瘤细胞、成纤维细胞及肝细胞.研究材料的细胞相容性,发现多种动物细胞在盐沥滤法所制备的丝素多孔支架材料上能够很好地黏附和增殖.通过调整丝素水溶液的浓度、溶解丝素溶剂体系、NaCl添加量和NaCl粒径,制备出结构和性质可以调控的三维支架.该结果为丝素多孔支架材料的制备提供了新的研究思路和方法.     

新型姜黄素结构衍生物抗肝癌细胞增殖作用的研究

采用MTT法对姜黄素结构衍生物（CCM系列化合物）进行抗人肝癌细胞Bel-7402和SMMC-7721活性筛选;利用流式细胞技术和荧光显微镜检测SMMC-7721细胞凋亡及周期分布;采用Western-Blot方法检测SMMC-7721中蛋白caspase-3和剪切后p17的表达.结果表明,化合物CCM-5和CCM-14抗肿瘤活性较好,其对SMMC-7721细胞的凋亡作用呈剂量依赖性,且凋亡率与阴性对照组相比有显著差异（P<0.01）;化合物浓度增高时,G0/G1期细胞减少,S期以及G2/M期细胞增加,凋亡峰SubG1峰增大;两个化合物均可增强caspase-3的表达,随着浓度的提高,caspase-3的表达趋势减弱,而剪切形式p17亚基表达量逐渐提高.因此,姜黄素结构衍生物CCM-5和CCM-14能抑制人肝癌细胞SMMC-7721细胞的增殖,促进凋亡,其作用机制可能与化合物诱导caspase-3活性的增强有关.