Survivin特异性siRNA诱导胰腺癌细胞凋亡的研究

目的 观察survivin特异性siRNA对胰腺癌细胞株Panc-1细胞增殖和凋亡的影响.方法 体外构建Survivin特异性siRNA表达载体p-Survivin-siRNA,转染胰腺癌细胞株Panc-1细胞系,RT-PCR和Western印迹法检验其对胰腺癌细胞株Panc-1细胞的RNA干扰(RNAi)效果.采用MTT法分析其对细胞增殖的影响,流式细胞仪检测其对细胞周期的影响.同时,采用Western印迹法检测半胱氨蛋白酶 3(caspase 3)的表达变化.结果 p-survivin-siRNA表达质粒高效而特异地剔降胰腺癌细胞株Panc-1细胞中survivin的表达,抑制肿瘤细胞增殖(P<0.01),阻断胰腺癌细胞株Panc-1细胞在G1期.随着survivin基因被沉默,caspase 3表达升高(P<0.01).结论 靶向survivin基因的siRNA表达载体可以显著抑制胰腺癌细胞株Panc-1细胞增殖,促进细胞凋亡.     

线粒体与细胞凋亡

凋亡是机体细胞的一种程序性死亡。线粒体作为细胞能量代谢的场所,不仅能诱导细胞凋亡,还是细胞凋亡的执行者。线粒体内的细胞色素C和细胞凋亡诱导因子参与凋亡的诱导,同时Caspase3、7、9还参与线粒体/细胞色素C诱导的细胞凋亡机制,线粒体内的Bc l-2和Bc l-X1也参与细胞凋亡的调节。对线粒体生命代谢活动的了解及运用对我们认识机体发生、发育及衰老有重要意义。     

细胞凋亡

细胞凋亡是一种自主性的细胞主动死亡，其触发因素众多，提示内部存在着不同的启动机制和运行通路；细胞凋亡与程序性细胞死亡二者各自蕴含的“程序性”具有不同的内涵，这两个概念有必要相互独立地使用下去。细胞凋亡是机体的一种正常生理机制，生理状态下参与调节体内正常细胞的更新和异常细胞的清除，而在病理状态下异常的凋亡又与肿瘤等多系统疾病的发生发展密切相关。     

重组Caspase-3基因的构建及其诱导胰腺癌细胞凋亡的观察

通过对Caspase—3(半胱氨酸蛋白酶3)大亚基(LS)，小亚基(SS)的重组，构建重组型Caspase—3基因(r—caspase—3)，并探讨r—Caspase—3基因诱导胰腺癌细胞凋亡的可能性，以寻求肿瘤基因治疗的新途径。采用分子生物学方法克隆Caspase—3的大、小亚基，体外进行重新排列组合，使大、小亚基原来的排序颠倒，构建出pcDNA3．1( )／r—Caspase—3真核表达质粒；利用脂质体瞬时转染人胰腺癌细胞PC—Ⅰ，RT—PCR检测r—Caspase—3mRNA的表达；流式细胞技术(FCM)检测转染胰腺癌细胞凋亡状况。结果显示：Caspase—3的大、小亚基被完整克隆，r—Gas—pase—3基因测序结果证实小亚基位于大亚基之前；RT—PCR扩增出894bp大小片段，流式细胞检测可见明显的凋亡峰出现。以上结果表明，重组r—Caspase—3基因其mRNA可在胰腺癌细胞中表达并自催化诱导细胞凋亡，可作为胰腺癌基因治疗的目的基因。     

胰腺癌细胞外泌体通过激活线粒体凋亡途径促进β细胞凋亡

目的:探讨胰腺癌细胞分泌的外泌体(exosome)对胰岛β细胞存活率和功能的影响及作用机制。方法:采用外泌体提取试剂盒提取小鼠胰腺癌细胞Pan02和MPC-83上清液外泌体,经磷钨酸染色后于透射电镜下鉴定形态;外泌体经荧光标记后与小鼠胰岛瘤MIN6细胞共孵育48 h,检测外泌体分泌水平和MIN6细胞的摄取水平;MTT和葡萄糖刺激的胰岛素分泌(GSIS)实验分别检测各组细胞的存活率和胰岛素分泌功能;q PCR检测微小RNA-204(miR-204)和Bcl-2 mRNA的表达;Western blot检测线粒体凋亡信号通路相关蛋白Bcl-2、Bax、caspase-3和细胞色素C(Cyt-C)的表达。结果:透射电镜结果显示2种胰腺癌细胞均能分泌外泌体,且Pan02细胞分泌更多。荧光标记的外泌体与胰岛β细胞共孵育结果显示,β细胞能够大量摄取胰腺癌细胞分泌的外泌体。MTT和GSIS实验结果显示,外泌体处理组的MIN6细胞存活率和高糖刺激的胰岛素分泌量显著低于未处理组(P0.01)。q PCR结果显示胰腺癌细胞分泌的外泌体富含miR-204,且外泌体处理后的MIN6细胞内Bcl-2的mRNA表达显著下调(P0.01)。Western blot结果显示,外泌体处理的MIN6细胞内Bcl-2蛋白表达显著下调(P0.05),Bax、cleaved caspase-3和Cyt-C蛋白表达显著上调(P0.01)。结论:胰腺癌细胞能够分泌外泌体,且该外泌体能够被胰岛β细胞摄取。胰腺癌细胞分泌的外泌体可以降低β细胞存活率和β细胞胰岛素的分泌功能,其机制可能通过外源性上调β细胞内miR-204的表达,进而抑制Bcl-2的mRNA和蛋白表达,最终激活β细胞内线粒体凋亡信号通路。     

免疫系统中细胞的程序性死亡：文献综述

细胞的程序性死亡是一种生理性死亡。近年来这种过程在免疫系统的发生与发展中也起着很重要的作用。影响免疫细胞程序性死亡的因素多种多样。本文简要综述了近年来在这方面的研究进展。     

凋亡抑制蛋白XIAP和促凋亡因子Smac在胰腺癌化疗抵抗中的作用

目的探讨x-相关凋亡抑制蛋白（XIAP）和促凋亡因子Smac在胰腺癌细胞化疗抵抗中的作用，以及转染胞浆表达型Smac基因靶向下凋XIAP对化疗药物诱导的胰腺癌细胞凋亡的影响。方法应用流式细胞术检测顺铂、5-FU介导的Panc-1、BXPC-3的凋亡率及胞浆染色分析细胞XIAP表达变化，Western blot分析XIAP、Smac、Caspase-3表达水平；构建pEGFP-NI／Smac真核表达载体并转染胰腺癌Panc-1细胞，流式细胞术检测转染Smac基因前后Panc-1细胞的凋亡敏感性。结果与BXPC-3细胞相比，Panc-1对顺铂或5-FU介导的凋亡具有较强抵抗性，Western blot分析显示Panc-1细胞高表达XIAP，在化疗药物作用下化疗敏感细胞BXPC-3胞浆内XIAP水平下降明显多于Panc-1细胞，而且凋亡的BXPC-3细胞释放入胞浆内的成熟Smac蛋白水平明显高于Panc-1细胞。转染胞浆表达型Smac基因至化疗抵抗Panc-1细胞，可明显下调其XIAP表达水平，促进效应Caspase-3分子活化，显著提高顺铂、5-FU诱导的细胞凋亡率。结论胰腺癌细胞XIAP的表达水平下调与其化疗敏感性有关，XIAP是克服化疗抵抗的重要靶分子，而上调Smac活性蛋白的胞浆表达作为一种有效调节信号，通过拮抗XIAP的凋亡抑制作用协同化疗药物促进胰腺癌细胞凋亡。     

bcl—2基因家族与细胞程序性死亡

ｂｃｌ－２家族是一类基因结构相似的参与细胞程序性死亡或称之为凋亡调节的基因，其成员可分为两类，一类能促进细胞的程序性死亡，一类能抑制细胞的程序性死亡。家族中各成员间可形成同源或异源二聚体，共同调节细胞内死亡和存活信息的平衡状态，最终决定细胞的命运。     

细胞凋亡与肿瘤

细胞凋亡是机体细胞衰老与死亡过程的一种主要形式。许多癌基因与抑癌基参与细胞凋亡过程，许多研究表明肿瘤的发生与细胞凋亡有关，对细胞凋亡的深入研究将为肿瘤的防治开辟新的途径     

生物材料诱导细胞凋亡的研究进展

针对近些年生物材料诱导细胞凋亡的现象和机制的研究情况，从凋亡信号传导角度分类综述了生物材料诱导细胞凋亡的途径，包括传统信号传导途径、死亡受体途径、以及近年发现的线粒体途径，另外也论述了生物材料诱导活性氧产生而发生的凋亡和影响细胞黏附导致的细胞凋亡等途径，发现生物材料诱导的细胞凋亡产生的途径具有明显的多样性、交叉性和多途径同时作用的特点。本综述对于生物材料诱导细胞凋亡机制的深入研究特别是生物材料的研制具有重要的指导意义。此外也为生物材料广泛应用于人类疾病特别是应用于与细胞凋亡紧密相关的癌症治疗提供了借鉴。     

BHLHB2 在人胰腺癌细胞株SU86.86的生物学功能研究

 【摘要】目的：进一步研究前期实验结果证明的人胰腺癌新颖的独立预后因子BHLHB2(cAMP -inducible basic helix-loop-helix domain containing, class B, 2) 在人胰腺癌细胞中的相关分子生物学功能。方法：应用siRNA干扰技术,在人胰腺癌细胞株SU86.86中使BHLHB2表达沉寂,然后进行迁徙实验,研究BHLHB2与细胞迁徙的关系;应用更生霉素（Actinomycin D,ACT-D)诱导细胞凋亡,研究BHLHB2与细胞凋亡的关系;应用Western Blot方法研究BHLHB2与EMT的相关性。结果: 人胰腺癌细胞BHLHB2 表达沉寂后,细胞的迁徙明显增快。应用ACT-D进行凋亡诱导,BHLHB2表达沉寂后、发生凋亡的细胞数目明显增加(10ng/ml, 增加 1.32倍, p<0.05; 100ng/ml, 增加 1.41倍, p<0.05)。Western Blot结果表明, BHLHB2表达沉寂后可显著降低E-cadherin的表达(降低 11.35 倍, p<0.01)。结论: BHLHB2 具有多种生物学功能,与人胰腺癌细胞的迁徙、细胞凋亡及EMT密切相关。     

Bafilomycin A1 induces apoptosis in the human pancreatic cancer cell line Capan-1

Bafilomycin A₁, a specific inhibitor of vacuolar type H⁺-ATPase, can inhibit the growth of a variety of cultured cells in a dose-dependent manner, but its mechanism is unclear. The aim of this study was to examine whether bafilomycin A₁ inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis. The effect of bafilomycin A₁ on tumour growth in vitroand in vivowas examined using an MTT assay and an in vivotumour model. The presence or absence of apoptosis was determined by morphology and DNA analysis of tumour cells. The concentration of bafilomycin A₁ for 50 per cent inhibition of cell viability during 72 h by the MTT assay was 5 nm. In DNA analysis, a ladder of fragmented DNA was detected in Capan-1 cells treated with bafilomycin A₁ at concentrations greater than 10 nm for 24 h. Nude mice bearing a xenografted Capan-1 cell line tumour received 4 weeks of bafilomycin A₁ (1·0 mg/kg per day). This treatment significantly inhibited tumour growth compared with controls after 21 days (P<0·05). Histopathological examination of tumour cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. These observations suggest that bafilomycin A₁ inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis. © 1998 John Wiley & Sons, Ltd.     

The effect of acute running and cycling exercise on T cell apoptosis in humans: A systematic review

This review analyses the influence of acute running and cycling exercise on T lymphocyte apoptosis. We included randomized controlled trials (RCTs) and non-randomized case–control studies (NRCTs) measuring apoptosis by flow cytometry. Cochrane Library, Scopus, PubMed and Ovid were searched for running and cycling intervention studies. Risk of bias was assessed by Cochrane Collaboration's tools. We included five NRCTs and one RCT with a total of 93 participants. The RCT found a higher percentage of apoptotic T helper cells identified by upregulation of Annexin V, caspase-3 and caspase-9 under hypoxic conditions, and only one NRCT reported a higher percentage of highly differentiated apoptotic T cells immediately after exercise. Three hours after exercise, the same NRCT showed an increase in several T cell subsets such as T helper, cytotoxic T, low differentiated and regulatory T cells. The interventions were very heterogeneous by exercise protocol and external conditions. High risk of bias in NRCTs restricts accuracy of the included studies. Imprecision due to the small sample size limits further evidence. In the future, scientists should include apoptotic measures into their research design, plan RCTs, measure apoptosis at different time points post-exercise and increase sample size.     

Elevations in cytosolic free Ca2+ are not required to trigger apoptosis in human leukaemia cells.

Previous studies have indicated that Ca2+ is a trigger for apoptosis (programmed cell death) in thymocytes and related cell lines. Recently we have shown that levels of apoptosis in leukaemic cells are diminished in Ca(2+)-deficient conditions, indicating that Ca2+ may be important in the mechanism of apoptosis in these cells. In the present study we investigated the possibility that Ca2+ serves as a trigger for apoptosis in the human leukaemic cell line, HL-60. Using fura-2 to measure cytosolic free Ca2+ concentrations, [Ca2+]i, in cell suspensions, and by using ratio imaging of fura-2 in single cells, we did not observe an early significant increase in [Ca2+]i in HL-60 cells undergoing apoptosis. The latter stages of apoptosis were, however, accompanied by increasing [Ca2+]i; these increases were apparently a result of, rather than a cause of, apoptosis. Furthermore, apoptosis could be induced in HL-60 cells under conditions of vastly reduced [Ca2+]i achieved by loading these cells with fura-2 in the presence of EGTA. These results indicate that elevation of [Ca2+]i is not a prerequisite for apoptosis in HL-60 cells and that apoptosis can occur in these cells in the presence of low [Ca2+]i.     

Aspergillus fumigatus conidia inhibit tumour necrosis factor- or staurosporine-induced apoptosis in epithelial cells

A major innate immune response to inhaled conidia of the opportunistic pathogen Aspergillus fumigatus (Af) is the synthesis of pro-inflammatory cytokines, which include tumour necrosis factor (TNF)-alpha, a known inducer of apoptosis. Modulation of host cell apoptosis has been reported to be one of the mechanisms whereby pathogens overcome host cell defences. Our study was designed to investigate whether or not Af conidia could modulate apoptosis induced by TNF-alpha or staurosporine (STS). Exposure of epithelial cells treated by these inducers and exposed to Af conidia decreased the number of apoptotic cells detected by Annexin V staining, analysis of nuclear morphology, terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end-labelling reaction and immunoblotting. Inhibition of apoptosis by Af conidia was seen in cells of the A549 pneumocyte II line, human tracheal epithelial 16HBE and primary human respiratory cells. Inhibition of apoptosis by Af conidia was also observed when apoptosis was induced by co-cultivating A549 cells with activated human alveolar macrophages. Unlike Af conidia, conidia of Cladosporium cladosporioides as well as latex beads or killed Af conidia have no inhibitory effect on TNF-alpha or STS-induced apoptosis. For TNF-induced apoptosis, the observed anti-apoptotic effect of Af conidia was found to be associated with a significant reduction of caspase-3.     

Cell death by apoptosis in acute leukaemia

We have previously demonstrated that when freshly isolated childhood T-cell acute lymphoblastic leukaemia cells are incubated in growth medium after isolation from blood, chromatin is rapidly cleaved into nucleosomal sized fragments that are multiples of 200 bp. The fragmentation is similar to that observed in other types of cells undergoing apoptosis or programmed cell death. In this study we describe a more comprehensive approach to the study of DNA fragmentation in leukaemia. Fragmentation was observed in freshly isolated cells from patients with T-cell acute lymphoblastic leukaemia and in one with common acute lymphoblastic leukaemia. Frozen samples of T-cell acute lymphoblastic leukaemia, common acute lymphoblastic leukaemia, and acute myeloid leukaemia cells also showed fragmentation of DNA. However, no fragmentation was evident in normal leukocytes treated under the same conditions. Ultrastructural studies on the isolated leukaemia cells demonstrate that the chromatin cleavage observed biochemically is associated with morphological changes characteristic of apoptosis.     

Role of epidermal growth factor gene in the development of pancreatic cancer and efficiency of inhibitors of this gene in the treatment of pancreatic carcinoma

The expression of epidermal growth factor receptors in normal and tumor cells of the pancreas, the type and incidence of EGFR gene polymorphism were studied. EGFR gene expression in pancreatic adenocarcinoma cells significantly surpassed that in normal pancreatic cells. On the other hand, AA genome and A allele polymorphism in the EGF gene nucleotide pair G-A 61 is a significant risk factor for pancreatic cancer. The effect of AG-1478 preparation (a new-generation inhibitor of EGFR) on apoptosis and cell proliferation in pancreatic cancer was evaluated. This preparation is not inferior to 5FU by its apoptotic effect and significantly reduces cell proliferation, its antiproliferative effect being 1.5 times higher than that of 5FU. __________ Translated from Kletochnye Tehnologii v Biologii i Meditsine, No. 2, pp. 106–109, April, 2008     

Anti-retroviral therapy reverses HIV-associated abnormalities in lymphocyte apoptosis

The objective of this study was to assess the role of anti-retroviral therapy (ART) on the susceptibility of peripheral blood lymphocytes (PBL) from HIV-1-infected individuals to activation-induced apoptosis and in comparison with changes in CD4 lymphocyte counts. Eleven symptomatic HIV⁺ patients were studied. Ex vivo apoptosis was measured in phytohaemagglutinin (PHA)-stimulated PBL and CD4 subsets by flow cytometry, at baseline and after 1 month (4–6 weeks) and 2/3 months of ART. Six patients had extended studies of the effects of therapy to a maximum of 21 months. Lymphocyte apoptosis was significantly elevated in HIV⁺ patients at baseline (median 22% compared with 7.5% in HIV^? risk-matched controls; P < 0.05). This decreased to control levels on ART (7.4% at 4–6 weeks, P < 0.01, and 6.2% at 8–12 weeks, P < 0.05, compared with baseline). Similar changes occurred in the CD4⁺ subpopulation. The decrease in apoptosis was maintained for several months, but the effect was rapidly lost if ART was discontinued. CD4 counts showed a reciprocal relationship to changes in apoptosis. The association of changes in apoptosis with those in CD4 counts suggests a link between programmed cell death and lymphocyte depletion. Apoptosis reduced in some individuals without any reduction in viral load, suggesting apoptosis may be influenced by factors in addition to the overall extent of HIV replication.     

Differential expression of clusterin in inducible models of apoptosis.

Apoptosis (programmed cell death) and necrosis can be readily distinguished morphologically and biochemically. The most striking biochemical change observed in apoptotic cells is the cleavage of the genomic DNA into discrete nucleosome sized fragments, producing a laddering pattern when the DNA is examined electrophoretically. It has recently been shown that RNA and protein products of the testosterone-repressed prostate message-2 gene are induced, coordinate with the onset of cell death. This gene has been isolated from a variety of species and tissues, it is highly conserved, and collectively referred to as clusterin. We have examined a number of inducible leucocyte models of apoptosis, including glucocorticoid and calcium ionophore induced thymocyte death, 'aged' neutrophils and cytotoxic T cells, and found that in these situations that cell death is not associated with up-regulation of clusterin gene expression. The finding that clusterin is not expressed in all cells undergoing apoptosis would suggest that this molecule is not critical to the mechanism of cell death. It does, however, provide the first example of a readily detectable marker which is differentially expressed in cells undergoing apoptosis and adds further weight to the argument that apoptosis is not a uniform phenomena, but is dependent on the nature of the cells involved and the means of induction.     

miR-671-5p通过靶向肿瘤坏死因子α诱导蛋白8调控胰腺癌细胞增殖和凋亡

目的：探讨miR-671-5p 靶向肿瘤坏死因子α 诱导蛋白8（ TNFAIP8）对胰腺癌细胞增殖和凋亡的影响。 方法：实时荧光定量PCR（ qRT-PCR）和免疫印迹检测20 例胰腺癌组织和与其配对的癌旁正常组织miR-617-5p、 TNFAIP8 mRNA和TNFAIP8表达水平,验证miR-671-5p 和TNFAIP8的靶向调控关系。将体外培养胰腺癌细胞 capan-1分为miR-NC组、miR-671-5p 组、si-NC 组、si-TNFAIP8 组、miR-671-5p+pcDNA组和miR-671-5p+pcDNATNFAIP8 组。采用四甲基偶氮唑蓝（ MTT）实验检测细胞活力,流式细胞术检测细胞凋亡,免疫印迹检测细胞周 期蛋白D1（ cyclin D1）、p21、B细胞淋巴瘤/ 白血病-2（ Bcl-2）和Bcl-2 相关蛋白（ Bax）的表达水平。结果：与 癌旁正常组织比较,胰腺癌组织miR-617-5p 表达量显著降低,TNFAIP8 的表达量显著升高。miR-671-5p 靶向负向 调控TNFAIP8 表达。与miR-NC组比较,miR-671-5p 组capan-1 细胞活力显著降低,细胞凋亡率显著升高,cyclin D1和Bcl-2 的表达量显著降低,p21 和Bax 的表达量显著升高;与si-NC 组比较,si-TNFAIP8 组capan-1 细胞活力 显著降低,细胞凋亡率显著升高,cyclin D1和Bcl-2 表达量显著降低,p21 和Bax 表达量显著升高;与miR-671- 5p+pcDNA 组比较,miR-671-5p+pcDNA-TNFAIP8 组capan-1 细胞活力显著升高,细胞凋亡率显著降低,cyclin D1和Bcl-2 的表达量显著升高,p21 和Bax 的表达量显著降低。结论：miR-671-5p 通过靶向下调TNFAIP8 抑制胰 腺癌细胞增殖并促进其凋亡。上调miR-671-5p 是胰腺癌潜在治疗靶点。