化疗药物诱导肾癌细胞凋亡的研究

目的：探讨化疗药物Cyrptophycin(CPP),长春新碱（VCR）,阿霉素（ADM）诱导肾癌细胞凋亡的时间效应与量效应关系。方法：采用HE染色,荧光染色,琼脂糖凝胶电泳,流式细胞术检测3种药物不同的药物浓度,不同作用时间所诱导的癌细胞凋亡的结果。结果：在一定的药物浓度上,用光镜,荧光显微镜,流式细胞仪均能观察到肾癌细胞凋亡的典型形态学改变,凝胶电泳证实有DNA裂解的现象,诱导凋亡的效率CPP＞VCR＞ADM,结论：抗肿瘤药CPP,VCR,ADM呵诱导肾癌细胞凋亡的发生,对指导临床肾癌的治疗有积极的意义。     

贫铀诱发细胞超微结构改变及DMSO的保护作用

目的观察贫铀(DU)对体外培养的人支气管上皮细胞超微结构的影响及二甲基亚砜(DMSO)的保护作用。方法DU作用人支气管上皮细胞(BEAS-2B)24 h后,用荧光染色法检测细胞存活率、坏死率和凋亡率,用透射电子显微镜(TEM)观察细胞超微结构改变。结果荧光显微分析表明,DU作用后,存活细胞数明显减少,凋亡和坏死细胞数显著增高,而DMSO对DU诱发的细胞凋亡和坏死有明显的保护作用。TEM显示,正常细胞和DMSO对照细胞整体形态、核质比例、各种细胞器及细胞骨架均结构清晰;DU处理的细胞,无论细胞内、外有无贫铀颗粒,均见细胞不同程度的凋亡或坏死,正常细胞结构改变或消失,特别是细胞内或外有DU颗粒的细胞,膜性细胞器改变明显,其他细胞器也观察到结构不清或空泡化;DMSO+DU组,即使细胞内外有贫铀分布,各种细胞器结构的改变也明显减轻,观察到有些线粒体、内质网等膜性结构发生膨胀,但细胞整体结构仍较清晰。结论DU可诱发细胞凋亡和坏死,并导致细胞超微结构改变,DMSO对DU所致细胞损伤有明显的保护效果。     

康莱特注射液诱发肾癌细胞凋亡及p53,bcl—2表达的研究

目的;探讨康莱特注射液抗肿瘤的作用机制。方法：利用ＭＴＴ法分析康莱特肾癌细胞的抑制作用,末端脱氧核苷酰转移酶法检测细胞凋亡,免疫组织化学法分析ｐ５３和ｂｃｌ－２基因表达的影响。结果：康莱特抑制肾癌细胞的ＩＣ５０为１９．３１μｌ／ｍｌ,５μｌ／ｍｌ,和１０μｌ／ｍｌ康莱特注射液具有诱发细胞凋亡的作用,细胞凋亡分别为３１．３０％和８９．７６％,康莱特浓度继续增加时,细胞凋亡的数量反而减少,１５μdispla     

细胞凋亡信号传导研究进展

细胞凋亡是受基因调控的一种主动性细胞自杀过程,它与细胞增殖的动态平衡是维系生长发育、内环境稳定和免疫调节所必须的最基本过程,细胞内有一整套极其复杂的信号系统精细地调控细胞凋亡过程,近年来人们对细胞凋亡信号传导途径的研究已取得明显进展,主要表现在:①蛋白酶Caspase家族在细胞凋亡中起重要作用;②线粒体内膜空间释放促死亡蛋白是导致凋亡发生的主要因素;③死亡配基和细胞内应激是细胞凋亡信号传导中两条重要途径。 1 Caspase家族在细胞凋亡中的作用 Caspase是特异酶切ASP氨基位点的半胱氨酸蛋白酶(Cysteinyl Aspartate Specific Proteinase),Caspase的发现来自线虫凋亡基因的遗传学研究,在哺乳动物以至少发现13个成员,根据其发现顺序统一命名为Caspase 1—13,目前尚不清楚为什么在哺乳动物中存在如此之多Caspase,而且发现在某些情况下,不同Caspase裂解相同的蛋白底物,推测可能原因有:①Caspase或许是以组织或亚细胞特异性方式激活的;②Caspase或许对底物具有专一性;③Caspase或许是按级联裂解顺序排列的;④Caspase为不同凋亡刺激所必须。 所有蛋白水解酶Caspase都具有相似的氨基酸序列、结构和底物特异性,它们均以酶原(Proenzyme)形式存在,由N端长副区(Prodomain)约30KD,大亚单位约2     

Cryptophycin诱导肾癌细胞凋亡的研究

目的 探讨诱导肾癌细胞凋亡的新方法及其机制。方法 采用ＨＥ滩色、荧光染色、琼脂糖电池等方法,分别对Ｃｒｙｔｏｐｈｙｃｉｎ（ＣＰＰ）不同的药物浓度（０,１,５,１０,５０ｐｍｏｌ／Ｌ）和不同增减时间段（４～２４ｈ）所诱导的凋亡细胞形态、出现时间和密度比例进行观察和比较。结果 在光镜下能观察到ＣＰＰ诱导的肾癌细胞凋亡的典型形态,用ＡＯ／ＥＢ荧光染色能区分出４种不同细胞,ＤＮＡ电泳证实有ＤＮＡ裂解的现象     

三氧化二砷诱导肝癌细胞凋亡的初步研究

目的：研究三氧化二砷（Ａｓ２Ｏ３）对肝癌细胞株Ｂｅｌ－７４０２的抑制增殖效应及凋亡诱导作用。方法：采用ＭＴＴ法检测药物对细胞的抑制作用、相差显微镜和电镜观察细胞形态变化、琼脂糖凝胶电泳测定ＤＮＡ Ｌａｄｄｅｒ、流式细胞术检测细胞周期变化。结果：各浓度Ａｓ２Ｏ３组（０．２５～１６μｍｏｌ／Ｌ）均可抑制肝癌细胞增殖,且抑制率具有浓度和时间依赖性,同作用时间呈直线相关,其中浓度组为１０μｍｏｌ／Ｌ）均可抑制肝癌细胞增殖,且抑制率具有浓度和时间依赖性,同作用时间呈直线相关,其中浓度组为１０μｍｏｌ／Ｌ抑制率最大,９６ｈ时达８３．８９％,２μｍｏｌ／Ｌ Ａｓ２Ｏ３以抑制肝癌细胞的增殖为主,但在形态及生化方面未见凋亡改变;１０μｍｏｌ／Ｌ Ａｓ２Ｏ３作用１０ｈ可见形态学变化,４８ｈ可见ＤＮＡ Ｌａｄｄｅｒ出现。流式细胞检查     

三氧化二砷诱导人卵巢癌细胞凋亡机制的研究

目的：探讨三氧化二砷（arsenic trioxide,As2O3)诱导人卵巢癌细胞株3AO细胞凋亡的机制。方法：以人卵巢癌细胞株3AO细胞为体外实验对象。通过台盼蓝染色法，测定不同浓度As2O3作用不同时间对3AO细胞的生长抑制率；采用流式细胞仪检测法，通过碘化丙（PI）／罗丹明123（Rhodammine 123,Rh123)双重染色测定3AO细胞线粒体跨膜电位（△ψm) ；通过吖啶橙染色，荧光显微镜观察As2O3作用后3AO细胞的形态变化。结果：As2O3对3AO细胞的生长具有明显的抑制作用，且具有时间与剂量依赖性（P＜0．05）；3.0μmol/L的As2O3作用48h后3AO细胞中PI－Rh123^-细胞与对照组相比，差异有显著性（P＜0．05）；As2O3作用后，3AO细胞呈现典型的凋亡细胞形态特征。结论：As2O3通过诱导凋亡来抑制人卵巢癌细胞株细胞的生长，其机制与线粒体跨膜电位△ψm下降有关。     

冬凌草甲素诱导食管癌细胞凋亡过程中线粒体结构与膜电位的改变

目的:研究冬凌草甲素(Oridonin,ORI)诱导食管癌细胞凋亡的过程中线粒体超微结构和功能的变化。方法:采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法和透射电镜法检测细胞凋亡和超微结构改变;罗丹明123(Rhodamine 123,Rho123)荧光探针标记流式细胞仪检测和分析线粒体跨膜电位(MTP,△Ψm)的改变。结果:32μg/ml ORI作用2h后电镜下SHEEC细胞线粒体增多,4h后线粒体肿胀空泡化、内部结构消失,8h后细胞核染色质成块状边集,细胞凋亡。ORI作用24h后,代表线粒体膜电位的Rho123荧光强度降低。结论:在ORI诱导下,SHEEC细胞线粒体有明显的形态和功能改变伴随线粒体△Ψm降低,线粒体改变可能是ORI诱导食管癌细胞凋亡过程的重要环节。     

法尼基转移酶抑制剂ManumyCin诱导HepG2细胞凋亡的研究

目的:研究法尼基转移酶抑制剂Manumycin对人肝癌HepG2细胞的抗肿瘤作用,并探讨其诱导凋亡的分子机制。方法:采用MTT(Methythiazolyltetrazolium)法观察法尼基转移酶抑制剂Manumycin对肝癌细胞株HepG2细胞增殖的抑制作用,荧光显微镜、DNA凝胶电泳、流式细胞术等技术检测细胞凋亡,应用Westernblot方法检测bcl-2、p53、bax的蛋白水平变化。结果:法尼基转移酶抑制剂Manumycin能明显抑制HepG2细胞的生长且呈浓度依赖性,其IC50为(17.65±0.58)μmol/L。荧光显微镜检查显示Manumycin处理的HepG2细胞DAPI染色后,细胞核内可见浓染致密的颗粒荧光,典型细胞可见新月型改变,固缩或片段化的核。DNA凝胶电泳可见典型的DNA梯形带。流式细胞DNA直方图上出现典型的亚二倍体“凋亡峰”,细胞凋亡与Manumycin作用的时间和浓度相关。Manumycin能时间依赖性地诱导HepG2细胞发生G2/M期阻滞。Manumycin处理HepG2细胞后,Westernblot检测结果显示p53蛋白表达明显增加,而bcl-2蛋白和bax蛋白表达无明显变化。结论:法尼基转移酶抑制剂Manumycin对人肝癌细胞株HepG2有强烈的细胞毒作用,其分子机制可能是诱导HepG2细胞凋亡。Manumycin诱导HepG2细胞凋亡与bcl-2蛋白和bax蛋白表达水平无关,而p53蛋白表达水平的上调可能在此过程中起了一定     

热疗对人宫颈癌Hela细胞凋亡的影响

目的:观察不同加温温度对人宫颈癌Hela细胞凋亡的影响.方法:人宫颈癌Hela细胞株常规方法培养,采用水浴加热法(温度为41℃、42.5℃、43.5℃、)对细胞进行加温处理,处理后继续培养24h.用流式细胞仪检测细胞凋亡,单细胞凝胶电泳(single cell gel electrophoresis,SCGE)法检测DNA受损状态.结果:随着温度的增加细胞凋亡率增加,42.5℃、43.5℃加温1h后细胞凋亡率最高分别为30.7%和34.6,坏死细胞分别为13.2%和29.6%.单细胞凝胶电泳发现42.5℃加温1h后40.0%的细胞有DNA损伤,43.5℃加温1h后80.0%以上的细胞DNA损伤,而41℃加温处理1h后仅有20.0%细胞DNA受损.结论:单独加温处理1h可诱导细胞凋亡,并导致细胞DNA损伤.     

Expression of Fas Antigen and Its Mediation of Apoptosis in Human Gastric Cancer Cell Lines

Fas, a member of the tumor necrosis factor receptor/nerve growth factor receptor family, induces apoptosis by crosslinking with Fas ligand or anti-Fas antibody in a variety of cultured cells. We examined the expression of Fas antigen and its mediation of apoptosis in six human gastric carcinoma cell lines. Flow cytometric analysis and western blotting revealed relatively high expression of Fas antigen in MKN-74 (wild-type p53 gene) and MKN-45 (wild-type), followed by MKN-1 (mutated), MKN-7 (mutated) and KATO-III (deleted). MKN-28 (mutated) showed minimal expression of the antigen. The expression was apparently enhanced by interferon-γ, except for MKN-1 and MKN-28. Anti-Fas antibody (100 ng/ml) induced nuclear fragmentation characteristic of apoptosis. Apoptosis occurred in a delayed fashion and the apoptotic index at 72 h was approximately 60% in MKN-74, 35% in MKN-45, and 20% in MKN-1 and KATO-III. A DNA ladder was noted in MKN-74 at 72 h. Expression levels of P53 and P21^Wafl did not change for up to 48 h in MKN-74. The biological effects did not correlate with endogenous Bcl-2 expression. These results indicated that a) Fas antigen is variably expressed in human cultured gastric carcinoma cells, b) the protein transduces an apoptotic signal which leads to delayed cell death, and c) susceptibility to the antibody correlates well with the expression level of Fas antigen.     

常规化疗药物诱导卵巢癌OVCAR-3细胞凋亡特点的分析

目的：观察抗卵巢癌药物顺铂、紫杉醇和阿霉素对卵巢癌细胞系ＯＶＣＡＲ－３体外生长和生存的影响并分析由它们所致的细胞死亡性质。方法：采用细胞形态学观察、细胞动力学检测及ＤＮＡ片段化分析等细胞和分子生物学方法,研究化疗药物对卵巢癌细胞的凋亡诱导作用。结果：上述药物在抑制ＯＶＣＡＲ－３细胞生长的同时可不同程度地诱导细胞凋亡。其中,紫杉醇诱导细胞凋亡的能力最强;较低剂量紫杉醇（１０＾－８Ｍ）和顺铂（２μｇ／     

ALEX1 Regulates Proliferation and Apoptosis in Breast Cancer Cells

Background: Arm protein lost in epithelial cancers, on chromosome X (ALEX) is a novel subgroup withinthe armadillo (ARM) family, which has one or two ARM repeat domains as opposed to more than six-thirteenrepeats in the classical Armadillo family members. Materials and Methods: In the study, we explore the biologicalfunctions of ALEX1 in breast cancer cells. Overexpression of ALEX1 and silencing of ALEX1 were performedwith SK-BR3 and MCF-7 cell lines. Cell proliferation and colony formation assays, along with flow cytometry,were carried out to evaluate the roles of ALEX1. Results: ALEX1 overexpression in SK-BR3 breast cancer cellsinhibited proliferation and induced apoptosis. Furthermore, depletion of ALEX1 in MCF-7 breast cancer cellsincreased proliferation and inhibited apoptosis. Additional analyses demonstrated that the overexpression ofALEX1 activated the intrinsic apoptosis cascades through up-regulating the expression of Bax, cytosol cytochromec, active caspase-9 and active caspase-3 and down-regulating the levels of Bcl-2 and mitochondria cytochrome c.Simultaneouly, silencing of ALEX1 inhibited intrinsic apoptosis cascades through down-regulating the expressionof Bax, cytosol cytochrome c, active caspase-9, and active caspase-3 and up-regulating the level of Bcl-2 andmitochondria cytochrome c. Conclusions: Our data suggest that ALEX1 as a crucial tumor suppressor genehas been involved in cell proliferation and apoptosis in breast cancer, which may serve as a novel candidatetherapeutic target.     

survivin 反义核酸诱导HeLa 细胞凋亡的实验

 目的　探讨survivin 反义核酸诱导HeLa 细胞发生凋亡的可能性,揭示该凋亡发生与Bcl22 和 Bax 之间的关系。方法　采用TUNEL 染色法,研究survivin 反义核酸与HeLa 细胞凋亡的关系;通过免 疫组织化学法检测凋亡相关基因Bcl22 和Bax 的表达。结果　survivin 反义核酸在体外能诱导HeLa 细 胞凋亡,下调Bcl22 的表达,增强Bax 的表达。结论　诱导HeLa 细胞凋亡是survivin 反义核酸抗HeLa 作用的机制之一,survivin 反义核酸可能通过下调Bcl22 的表达,增强Bax 的表达,诱导HeLa 细胞发生 凋亡。     

Cellular Effect of Curcumin and Citral Combination on Breast Cancer Cells: Induction of Apoptosis and Cell Cycle Arrest

Purpose

The unmanageable side effects caused by current chemotherapy regimens to treat cancer are an unresolved problem. Although many phytonutrients are useful as chemoprevention without side effects, their effects are slower and smaller than conventional chemotherapy. In the present work, we examined the cumulative effect of two phytonutrients, curcumin and citral, on breast cancer cell lines and compared their effect with the known chemotherapy regimen of cyclophosphamide, methotrexate, and 5-fluorouracil.

Methods

Using cultured breast cancer and normal epithelial cells, the cytotoxic and apoptotic effect of curcumin and citral was evaluated in vitro. The synergistic effect of curcumin and citral was calculated by a combination index study using the method by Chou and Talalay. Cell death pathways and mechanisms were analyzed by measuring intracellular reactive oxygen species (ROS) and apoptotic protein levels.

Results

Curcumin and citral caused dose and time dependent cell death and showed a synergistic effect at effective concentration EC₅₀ and above concentrations in breast cancer cells without disturbing normal breast epithelial cells. With combination curcumin and citral treatment, apoptosis induction and cell cycle arrest at G0/G1 phase in breast cancer cells were observed. Curcumin and citral generated ROS and activated p53 and poly (ADP-ribose) polymerase-1 mediated apoptotic pathways.

Conclusion

The results of this study suggest that curcumin and citral in combination may be a useful therapeutic intervention for breast cancer.     

Fenugreek Induced Apoptosis in Breast Cancer MCF-7 Cells Mediated Independently by Fas Receptor Change

Trigonella foenum in graecum (Fenugreek) is a traditional herbal plant used to treat disorders like diabetes,high cholesterol, wounds, inflammation, gastrointestinal ailments, and it is believed to have anti-tumor properties,although the mechanisms for the activity remain to be elucidated. In this study, we prepared a methanol extractfrom Fenugreek whole plants and investigated the mechanism involved in its growth-inhibitory effect on MCF-7 human breast cancer cells. Apoptosis of MCF-7 cells was evidenced by investigating trypan blue exclusion,TUNEL and Caspase 3, 8, 9, p53, FADD, Bax and Bak by real-time PCR assays inducing activities, in thepresence of FME at 65 μg/mL for 24 and 48 hours. FME induced apoptosis was mediated by the death receptorpathway as demonstrated by the increased level of Fas receptor expression after FME treatment. However,such change was found to be absent in Caspase 3, 8, 9, p53, FADD, Bax and Bak, which was confirmed by atime-dependent and dose-dependent manner. In summary, these data demonstrate that at least 90% of FMEinduced apoptosis in breast cell is mediated by Fas receptor-independently of either FADD, Caspase 8 or 3, aswell as p53 interdependently.     

MiR-218-5p通过靶向抑制TDP1表达促进鱼藤酮诱导的胃癌细胞凋亡

目的 探讨过表达miR-218-5p和抑制TDP1的表达对鱼藤酮诱导损伤胃癌细胞凋亡的影响,阐明其可能的作用机制。方法 采用RT-PCR检测人正常胃黏膜上皮细胞和四种胃癌细胞中miR-218-5p及TDP1表达水平,并分析其相关性。双荧光素酶报告基因验证miR-218-5p对TDP1的靶向调控作用。采用1.0 μmol/L鱼藤酮诱导胃癌细胞损伤,流式细胞术检测细胞周期及凋亡率。Western blot检测细胞线粒体中TDP1水平及细胞Bax、Cyt-c蛋白的表达。结果 miR-218-5p在胃癌细胞中低表达（P<0.05）,TDP1高表达（P<0.01）,两者表达呈负相关（R²=0.9580, P=0.0212）。与对照组比较,损伤组SGC-7901细胞发生G1期阻滞,凋亡率升高（P<0.01）。与损伤组比较,miR-218-5pmimic组SGC-7901细胞G1期阻滞加剧,细胞凋亡率进一步升高（P<0.01）,Bax及Cyt-c表达上调（P<0.01）,而线粒体中TDP1蛋白水平降低（P<0.01）;TDP1过表达组细胞G1期阻滞得到缓解,凋亡率降低（P<0.01）,线粒体中TDP1蛋白水平升高（P<0.01）,Bax及Cyt-c表达降低（P<0.01）。结论 miR-218-5p可靶向抑制TDP1表达,诱导胃癌细胞凋亡,其作用机制可能与抑制线粒体DNA损伤修复及功能维持、激活线粒体内源性凋亡途径有关。     

右美托咪定对顺铂诱导的非小细胞肺癌细胞凋亡及氧化应激的影响

目的 探讨右美托咪定(Dex)对顺铂诱导的人非小细胞肺癌(NSCLC)细胞凋亡的影响及其潜在机制.方法 将人非小细胞肺癌细胞系分为3组:对照组、顺铂组和Dex组.采用Western Blot法检测各组细胞中凋亡相关蛋白、细胞色素C、mTOR/ERK1/2信号分子及E-钙粘蛋白的表达,采用试剂盒法检测活性氧(ROS)的含...