二烯丙基二硫上调Beclin1 介导白血病K562 细胞自噬性死亡的研究

目的：通过二烯丙基二硫诱导白血病K562 细胞发生自噬性死亡,探讨其作用机制。方法：40 mg/LDADS 作用K562 细胞12 小时后,透射电镜观察K562 细胞超微结构,MDC 染色荧光显微镜观察自噬泡及流式细胞仪定量检测自噬率,RT-PCR 检测Beclin1mRNA 的表达水平。结果：DADS 作用后的K562 细胞后,透射电镜可观察到胞质内出现大量自噬体;MDC染色荧光显微镜观 察显示,K562 细胞胞浆中的自噬泡明显增多,而空白组与溶媒组胞浆中的自噬泡很少;流式细胞术定量测定空白对照组、溶媒对 照组、DADS药物组自噬率分别为(7.27± 5.60)%、(7.10± 5.13)%、(27.39± 6.51)%（P<0.05）;空白对照组为0.658± 0.007,溶媒对 照组为0.671± 0.012,两者的Beclin1mRNA 的表达强度无明显差异（P>0.05）,DADS 药物组为0.911± 0.008,高于对照组（P<0. 05）。结论：二烯丙基二硫可诱导白血病k562 细胞发生自噬性死亡,其机制可能与Beclin1 的上调有关。     

二烯丙基二硫对人白血病K562细胞增殖及Bcl-2表达的影响

目的:探讨二烯丙基二硫(DADS)对白血病K562细胞增殖的影响,以及Bcl-2表达的调节作用.方法:用DADS预处理白血病K562细胞构建细胞模型,MTT法分别检测不同DADS浓度(5 gmL-1、10 g mL-1、20 g mL-1、40 g mL-1)和不同处理时间(6h、12h、24h、48h)细胞增殖情况,IC50浓度处理白血病K562细胞之后,Western blot和RT-PCR检测Bcl-2蛋白和mRNA表达水平.结果:MTT结果显示DADS能够抑制白血病K562细胞的增殖,且呈剂量和时间依赖性;IC50浓度的DADS处理K562细胞24小时后,Bcl-2蛋白和mRNA的表达量显著减少.结论:DADS可显著抑制K562细胞增殖,而这一作用可能与Bcl-2表达下调有关.     

二烯丙基二硫作用于K562细胞后凋亡相关基因的差异表达

目的:研究二烯丙基二硫(diallyl disulfide DADS)作用于人白血病K562细胞后凋亡相关基因的差异表达,为进一步探讨DADS诱导K562细胞凋亡的分子机制提供基础.方法:采用瑞士-吉姆萨和Hoechst33342染色观察细胞形态学变化.运用基因芯片技术检测40 mg/LDADS作用于K562细胞24h后凋亡相关基因的差异表达,选择其中上调基因GADD45A、下调基因HO-1运用RT-PCR技术进行验证.结果:40mg/L的DADS作用于K562细胞后出现凋亡所具有的典型形态学变化.40 mg/L DADS作用K562细胞24 h后有14个凋亡相关差异表达基因.GADD45A、HO-1基因表达情况与基因芯片结果一致.结论:DADS可能通过多个基因和多条信号转导通路共同作用诱导人白血病细胞K562凋亡.     

二烯丙基二硫诱导人白血病K562 细胞凋亡及其对Bax、Bag-1 表达的影响

目的:探讨二烯丙基二硫(Diallyl disulfide)诱导白血病K562细胞凋亡的作用及其机制。方法:采用吖啶橙/溴化乙啶(AO/EB)染色法观察细胞凋亡形态学变化;DNA琼脂糖凝胶电泳测定DNA梯度带;RT-PCR法检测BAG-1、BAX基因的mRNA表达变化。结果:DADS可诱导K562细胞凋亡。其对K562细胞的凋亡效用与药物浓度、有明显依赖关系;DNA琼脂糖凝胶电泳示:40mg/LDADS作用K562细胞48小时后能够产生明显的梯形电泳图谱(DNA ladder):DADS作用48h后,BAX mRNA表达水平较对照组上调;BAG-1 mRNA较对照组下调(差异具有统计学意义,P<0.05)。结论:DADS能够诱导K562细胞凋亡,其凋亡机制可能与上调BAX,下调BAG-1有关。     

沉默UVRAG基因在二烯丙基二硫诱导K562细胞中caspase3的表达

目的:沉默UVRAG基因在DADS诱导K562细胞中观察caspase3的表达。方法:以K562细胞为细胞模型,将构建成功并筛选出最有效干扰抑制UVRAG基因的si RNA序列片段采用lipofectamine TM2000脂质体转染法转染白血病K562细胞,组别为:空白对照组,转染试剂组、阴性对照组,阳性对照组,转染24小时后,利用QT-PCR检测UVRAG m RNA的表达水平以此观察干扰效果,再以40 mg/LDADS处理转染试剂组12小时,采用蛋白印迹(Western Blotting)技术检测凋亡相关基因caspase3的表达。结果:QT-PCR显示:与空白组相比,UVRAG m RNA表达明显减少,表明沉默UVRAG基因成功;Western Blotting显示:DADS处理的干扰成功的K562细胞12小时后,检测到caspase3的蛋白表达水平下降。结论:沉默UVRAG基因能使白血病K562细胞中凋亡相关基因caspase3的蛋白表达水平下降,提示抑制UVRAG表达的同时也可能抑制DADS诱导K562细胞的凋亡。     

DADS 上调K562 细胞p21WAF1 表达对细胞生长阻抑和凋亡 的实验研究

目的:探讨二烯丙基二硫(DADS)对体外培养的人白血病细胞系K562细胞生长阻抑和凋亡作用及机制。方法:采用MTT分析法检测细胞活性、流式细胞术分析细胞周期及凋亡率、免疫组化检测p21WAF1基因表达。结果:1).DADS在10mg/L～80 mg/L范围内,对K562细胞的抑制作用呈剂量-时间依赖效应;2).不同浓度DADS作用于K562细胞24h后,细胞周期发生了变化:DADS可以将K562细胞阻滞于G2/M期;3).DADS浓度在10mg/L～80mg/L时作用K562细胞24h后,凋亡率逐渐升高,有显著的统计学意义(P<0.05或P<0.01);4).用浓度分别为0mg/L,20mg/L,40mg/L,80mg/L处理K562细胞24h后,p21WAF1蛋白表达上调,有统计学意义(P<0.05或P<0.01),溶媒组和阴性对照组无差别(P>0.05)。结论:DADS有抑制K562细胞增殖和促进K562细胞凋亡的作用。其作用的可能机制与上调细胞周期蛋白依赖性激酶抑制剂p21WAF1表达,从而诱使k562细胞阻滞于G2/M期有关。     

神经胶质瘤U251细胞氧化损伤中自噬和凋亡过程的时间顺序

目的：探讨过氧化氢（H2O2）诱导神经胶质瘤U251细胞损伤中自噬和凋亡发生的时间顺序。方法：实验分为4组：正常对照组、1mmol／L H2O2作用（6h、12h、24h）组。应用MTF法检测H202对神经胶质瘤U251细胞生存率的影响;MDC染色检测自噬空泡的变化;流式细胞仪检测细胞凋亡率变化。Western blot检测Beclin1和胞浆cyt c蛋白的表达。结果：与对照组相比,1mmol／L H2O2作用下,U251细胞存活率明显降低,并呈时间依赖性。与对照组相比,1mmol／L H2O2作用后,6h时U251细胞自噬空泡明显增加,自噬相关蛋白Beclin1表达明显增加,12h、24h细胞自噬水平逐渐增强;而6h时未见细胞凋亡率明显变化及cyt c由线粒体向胞浆的释放,12h、24h时细胞凋亡率明显增加,胞浆中cyt c蛋白表达明显增强（P〈0．05）。结论：氧化损伤能够诱导神经胶质瘤U251细胞发生自噬和凋亡,并且自噬发生于凋亡之前。     

二烯丙基二硫通过激活Caspase3和释放Cyt-c诱导Raji细胞凋亡

二烯丙基二硫（diallyl disulfide,DADS）作为天然植物大蒜中的提取物,能抑制多种肿瘤细胞生长,但其抑瘤的分子机制还不十分清楚。在该研究中,作者采用CCK-8（cell counting kit）技术检测发现,DADS能有效地抑制人淋巴瘤Raji细胞增殖,形态学观察、DNA琼脂糖凝胶电泳和流式细胞仪检测证实DADS呈时间和浓度依赖性诱导Raji细胞凋亡, DADS处理细胞24 h后, MCL1和Bcl-2蛋白表达下降,而Bax 和Bak蛋白表达水平无变化,Bid和Caspase3被激活,线粒体中Cyt-c释放增多,用Caspase 抑制剂Z-VAD-FMK能部分阻断DADS诱导人淋巴瘤Raji细胞凋亡, 提示DADS诱导的Raji细胞凋亡作用通过Bcl-2/MCL1-线粒体-caspase3通路介导。     

RORα高表达对二烯丙基二硫抑制人胃癌细胞增殖与迁移侵袭的影响

目的观察高表达RORα对二烯丙基二硫(DADS)抑制人胃癌MGC803细胞增殖、迁移与侵袭的影响。方法集落形成实验与流式细胞术检测细胞增殖与细胞周期;细胞划痕和Transwell实验分别检测细胞迁移与侵袭。RT-PCR与Western blot分别检测RORα、MMP-9和TIMP3 mRNA与蛋白表达水平。结果RT-PCR与Western blot检测显示,RORα高表达与DADS处理较对照组与空载体组RORαmRNA与蛋白表达明显上调,DADS+RORα高表达组上调更为显著(P<0.05)。与对照组和空载体组比较,RORα高表达与DADS处理组MMP-9表达下调,TIMP3表达上调,DADS+RORα高表达组改变最为显著。集落形成实验显示,RORα高表达与DADS处理组较对照组与空载体组的集落形成率明显降低。流式细胞术显示,与对照组和空载体组比较,RORα高表达与DADS处理组G2/M期细胞比率明显升高。细胞划痕和Transwell实验显示,RORα高表达与DADS处理组细胞迁移与侵袭能力明显降低。结论RORα高表达可通过上调TIMP3与下调MMP-9促进DADS阻滞MGC803细胞G2/M期和抑制增殖与迁移侵袭。     

survivin在二烯丙基二硫诱导HepG2细胞凋亡中的作用

目的:研究survivin在二烯丙基二硫(diallyl disulfide,DADS)诱导HepG2细胞凋亡中的作用。方法:MTT法检测细胞的生长活性;流式细胞仪检测细胞周期;RT-PCR法检测survivin mRNA水平;Western blot法检测survivin蛋白水平。结果:用药组HepG2细胞活性与正常组相比,随着药物浓度的增加(25,50,100,200μmol/l),分别下降了9．3%、10．4%、21．6%、31．2%,HepG2细胞凋亡率分别增加0．83%、1．97%、6．0%、9．9%,低浓度(25,50μmol/l)的DADS可以诱导survivin mRNA和蛋白表达升高,而高浓度的(100,200μmol/l)DADS可以降低survivin mRNA和蛋白表达。结论:DADS诱导HepG2细胞survivin表达增加,抵抗DADS诱导HepG2细胞的凋亡作用。     

自噬抑制剂氯奎对低氧诱导肺动脉平滑肌细胞增殖的影响

目的：探讨自噬抑制刺氯喹（cQ）在低氧（hypoxia）调节肺动脉平滑肌细胞（PASMCs）增殖中的作用。方法：将体外培养的大鼠PASMCs分为4组：正常对照组、1％低氧组、50μmol／L氯喹＋1％低氧组、50ttmol／L氯喹组。MTF方法检测各组的PASMCs增殖率;MDC染色检测细胞自噬空泡的变化;Westernblot方法检测微管相关蛋白轻链3（LC3）蛋白的表达变化;划痕法检测细胞迁移的变化。结果：与对照组比较,氯喹组的PASMCs细胞增殖率无明显变化。与对照组比较,1％低氧组PASMCs增殖率明显增加,细胞内出现大量自噬空泡,细胞迁移速度明显增加。细胞LC3-Ⅱ蛋白表达增强。与1％低氧组比较,氯喹与低氧联合作用时细胞自噬空泡的积聚以及rE3．II蛋白表达增强,但细胞增殖率和迁移明显降低。结论：低氧激活自噬过程并促进了PASMCs增殖和迁移,而自噬抑制剂氯喹在一定程度上通过抑制自噬进程,达到抑制肺动脉平滑肌细胞增殖和迁移的作用。     

Proliferation of Toxoplasma gondii Suppresses Host Cell Autophagy

Autophagy is a process of cytoplasmic degradation of endogenous proteins and organelles. Although its primary role is protective, it can also contribute to cell death. Recently, autophagy was found to play a role in the activation of host defense against intracellular pathogens. The aims of our study was to investigate whether host cell autophagy influences Toxoplasma gondii proliferation and whether autophagy inhibitors modulate cell survival. HeLa cells were infected with T. gondii with and without rapamycin treatment to induce autophagy. Lactate dehydrogenase assays showed that cell death was extensive at 36-48 hr after infection in cells treated with T. gondii with or without rapamycin. The autophagic markers, LC3 II and Beclin 1, were strongly expressed at 18-24 hr after exposure as shown by Western blotting and RT-PCR. However, the subsequent T. gondii proliferation suppressed autophagy at 36 hr post-infection. Pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), down-regulated LC3 II and Beclin 1. The latter was also down-regulated by calpeptin, a calpain inhibitor. Monodansyl cadaverine (MDC) staining detected numerous autophagic vacuoles (AVs) at 18 hr post-infection. Ultrastructural observations showed T. gondii proliferation in parasitophorous vacuoles (PVs) coinciding with a decline in the numbers of AVs by 18 hr. FACS analysis failed to confirm the presence of cell apoptosis after exposure to T. gondii and rapamycin. We concluded that T. gondii proliferation may inhibit host cell autophagy and has an impact on cell survival.     

Insulin withdrawal-induced cell death in adult hippocampal neural stem cells as a model of autophagic cell death

The term "autophagic cell death" was coined to describe a form of cell death associated with the massive formation of autophagic vacuoles without signs of apoptosis. However, questions about the actual role of autophagy and its molecular basis in cell death remain to be elucidated. We recently reported that adult hippocampal neural stem (HCN) cells undergo autophagic cell death following insulin withdrawal. Insulin-deprived HCN cells exhibit morphological and biochemical markers of autophagy, including accumulation of Beclin 1 and the type II form of microtubule-associated protein 1 light chain 3 (LC3) without evidence of apoptosis. Suppression of autophagy by knockdown of Atg7 reduces cell death, whereas promotion of autophagy with rapamycin augments cell death in insulin-deficient HCN cells. These data reveal a causative role of autophagy in insulin withdrawal-induced HCN cell death. HCN cells have intact apoptotic capability despite the lack of apoptosis following insulin withdrawal. Our study demonstrates that autophagy is the default cell death mechanism in insulin-deficient HCN cells, and provides a genuine model of autophagic cell death in apoptosis-intact cells. Novel insight into molecular mechanisms of this underappreciated form of programmed cell death should facilitate the development of therapeutic methods to cope with human diseases caused by dysregulated cell death.     

小豆蔻明调控自噬抑制卵巢癌SKOV3细胞增殖的研究

目的：卵巢癌为女性常见病,死亡率高,分子靶向治疗药物较少,研发高效低毒的靶向新药意义重大。细胞自噬（autophagy）维持着细胞内稳态和生长,是抗癌药物作用的关键靶部位。本课题旨在明确小豆蔻明（Cardamonin,CAR）调控自噬对卵巢癌SKOV3细胞增殖的影响。方法：体外培养卵巢癌SKOV3细胞,不同药物分组处理,荧光显微镜下观察单黄酰戊二胺（MDC）染色后细胞自噬囊泡,WesternBlot法检测细胞自噬相关蛋白LC3的表达,四氮唑蓝（MTT）法观察SKOV3细胞增殖情况,流式细胞术检测SKOV3细胞凋亡的变化。结果：自噬抑制剂3-MA显著性降低SKOV3细胞内MDC染色的荧光颗粒数目、LC3II蛋白表达,抑制细胞增殖;而CAR（高、中剂量）、雷帕霉素和AZD8055与3-MA联用后细胞内MDC荧光颗粒数目增多、LC3II蛋白表达增加、细胞增殖抑制率及凋亡率明显升高;且高剂量CAR（10^-6mol·L^-1）的作用比低剂量CAR（10^-6mol·L^-1）明显。结论：CAR能够抑制SKOV3细胞增殖,诱导细胞自噬,促进细胞凋亡。CAR有望成为卵巢癌药物治疗的先导化合物。本研究为进一步研发此类化合物提供了实验依据及一定的理论基础。     

Crosstalk between autophagy and apoptosis in the regulation of paclitaxel-induced cell death in v-Ha-ras-transformed fibroblasts

The previous studies by this author group has shown that paclitaxel, a mitotic inhibitor used in breast cancer chemotherapy, inhibits cell growth via induction of Raf-1-dependent apoptosis. In this article, the role of autophagy in paclitaxel anticancer action was investigated using v-Ha-ras-transformed NIH 3T3 cells. Paclitaxel induced a notable increase in the number of fluorescent particles labeled with monodansylcadaverine (MDC), a specific marker for autophagic vacuoles. MDC-labeled vacuoles clearly exhibited the fluorescent-tagged LC3 in cells transiently overexpressing GFP-LC3 (a protein that associates with autophagosome membranes). However, autophagy inhibition with 3-methyladenine (3-MA) failed to rescue v-Ha-ras-transformed NIH 3T3 cells from paclitaxel-induced cell death. More interestingly, the apoptosis inhibition by overexpression of the X-linked inhibitor of apoptosis (XIAP) did not fully block the cell death by paclitaxel, implying that apoptosis inhibition might accelerate the autophagic components of the paclitaxel response. Conversely, Raf-1 shRNA expression protected against paclitaxel-induced cell death through the simultaneous inhibition of both autophagy and apoptosis. These results suggest that both autophagy and apoptosis act as cooperative partners to induce cell death in v-Ha-ras-transformed NIH 3T3 cells treated with paclitaxel.     

DC-CIK对K562/A多药耐药基因mdr1表达的影响

目的：体外观察树突状细胞（dendritic cell,DC）联合细胞因子诱导的杀伤细胞（cytokine inducedkiller,CIK）对K562/A细胞株多药耐药基因mdr1表达的影响。方法：采集健康人的外周血,分离出单个核细胞（peripheral blood mononuclear cell,PBMC）,在体外加入多种细胞因子经诱导生成DC及CIK细胞,以流式细胞仪检测其表面标志,将DC细胞内加入K562/A细胞裂解物致敏后,再与CIK细胞混合培养48小时。将致敏后的DC-CIK细胞与K562/A及K562分组培养后以荧光定量PCR检测其mdr1基因表达的情况,PBMC作为对照组。结果：RT-PCR中可见K562/A＋DC-CIK组中mdr1 mRNA表达较K562/A明显降低,经荧光定量PCR观察到K562/A内mdr1 mRNA表达为K562的10.27倍、K562/A/PBMC略低于未处理的K562/A（P〉0.05）,K562/A/DC-CIK细胞中mdr1 mRNA含量较K562/A、K562/A/PBMC少（P〈0.05）。DC-CIK细胞与细胞株混合培养后,mdr1基因表达较混合培养前明显降低。结论：实验数据显示DC-CIK可使耐药细胞株内mdr1基因表达下调。但K562与DC-CIK混合培养后该基因降低不明显,提示该基因在细胞中存在着基础表达,意义在于维持细胞内稳态。目前针对逆转白血病耐药的研究较少,需要多进行相关研究以拓宽细胞免疫治疗在逆转耐药领域的应用。DC-CIK是具有发展潜力的抗肿瘤方法。本实验将为下一阶段研究逆转耐药的机制提供依据,DC-CIK细胞免疫疗法有望成为逆转肿瘤耐药的新方法。     

Autophagy and Cancer Therapy

Autophagy is a dynamic process of protein degradation which is typically observed during nutrient deprivation. Recently, interest in autophagy has been renewed among oncologists, because different types of cancer cells undergo autophagy after various anticancer therapies. This type of non-apoptotic cell death has been documented mainly by observing morphological changes, e.g., numerous autophagic vacuoles in the cytoplasm of dying cells. Thus, autophagic cell death is considered programmed cell death type II, whereas apoptosis is programmed cell death type I. These two types of cell death are predominantly distinctive, but many studies demonstrate cross-talk between them. Whether autophagy in cancer cells causes death or protects cells is controversial. In multiple studies, autophagy has been inhibited pharmacologically or genetically, resulting in contrasting outcomes—survival or death—depending on the specific context. Interestingly, the regulatory pathways of autophagy share several molecules with the oncogenic pathways activated by tyrosine kinase receptors. Tumor suppressors such as Beclin 1, PTEN, and p53 also play an important role in autophagy induction. Taken together, these accumulating data may lead to development of new cancer therapies that manipulate autophagy.