瘦素抑制H_2O_2诱导的大鼠心肌细胞凋亡

目的:观察瘦素(leptin)对H2O2诱导的大鼠心肌细胞凋亡的影响并探讨其作用机制。方法:应用脱氧三磷酸尿苷缺口末端标记(TUNEL)法观察瘦素对H2O2诱导的大鼠心肌细胞H9c2凋亡的影响;应用Western blotting法观察瘦素、H2O2对caspase-3、胞外信号调控激酶(ERK)活性的影响。结果:(1)瘦素对H2O2诱导的H9c2细胞凋亡具有显著的抑制作用(与对照组比较P0.01),该作用可被ERK激酶抑制剂PD98059所阻断。(2)H2O2明显抑制ERK活性;而瘦素可激活ERK并部分阻断H2O2诱导的caspase-3激活。结论:瘦素对H2O2诱导的H9c2细胞凋亡具有抑制作用,其机制可能与其激活ERK信号途径有关。     

奥沙利铂联合柔红霉素诱导结肠癌细胞系HT-29凋亡

目的研究奥沙利铂联合柔红霉素对结肠癌细胞凋亡的影响及机制。方法分别用不同浓度的奥沙利铂和柔红霉素处理结肠癌细胞HT-29,MTT法检测细胞增殖并计算半数抑制浓度。分别用半数抑制浓度的奥沙利铂、柔红霉素及二者联合处理HT-29细胞,MTT法测定细胞增殖;平板克隆实验检测细胞克隆形成;流式细胞计量术检测细胞凋亡;Western blot检测细胞中活化的caspase-3(cleaved caspase-3)、第10号染色体同源缺失性磷酸酶-张力蛋白(PTEN)、磷酸化Akt(p-Akt)蛋白表达。结果不同浓度的奥沙利铂和柔红霉素均可以抑制HT-29细胞增殖(P<0. 05),其半数抑制浓度为:柔红霉素约0. 23μmol/L,奥沙利铂约40 mg/L。柔红霉素、奥沙利铂和奥沙利铂联合柔红霉素处理后的HT-29细胞增殖能力、克隆形成能力降低;细胞凋亡率升高;细胞中cleaved caspase-3蛋白表达升高;细胞中p-Akt蛋白表达降低;PTEN蛋白表达升高。奥沙利铂联合柔红霉素作用高于单纯柔红霉素或奥沙利铂处理(P<0. 05)。结论奥沙利铂联合柔红霉素能够诱导结肠癌细胞HT-29凋亡,其作用机制可能与PTEN/Akt信号调控有关。     

苦参碱对人结肠癌HT-29细胞增殖、凋亡与侵袭的作用及机制研究

目的探讨苦参碱(MAT)对人结肠癌HT-29细胞增殖、凋亡与侵袭的影响及机制。方法将人结肠癌HT-29细胞分为空白对照组与苦参碱组(0.125、0.25、0.5、1mg/L)。不同浓度MAT干预24、48、72h后,MTT方法检测苦参碱对HT-29细胞增殖的抑制作用。1mg/L MAT干预48 h后,流式细胞术检测HT-29细胞的凋亡率;Transwell侵袭实验检测苦参碱对HT-29细胞侵袭能力的抑制作用;Western blot实验检测苦参碱对HT-29细胞Bcl-2、Bax、MMP-2与MMP-9的表达。结果不同浓度苦参碱均能显著抑制HT-29细胞增殖,且呈时间和浓度依赖性。1mg/L MAT干预48 h后,HT-29细胞凋亡率升高;侵袭能力降低(P0.01);Bax、MMP-2与MMP-9蛋白表达水平升高,Bcl-2蛋白表达水平降低(P0.01)。结论苦参碱能够抑制人结肠癌HT-29细胞增殖与侵袭,并促进凋亡。     

脂肪间充质干细胞条件培养基诱导结肠癌HT29细胞凋亡

目的:观察脂肪间充质干细胞条件培养基(ASC-CM)对结肠癌HT29细胞的增殖抑制作用。方法:将体外培养生长良好的HT29细胞分为两组,一组加入ASC-CM再培养(实验组),另一组加入DMEM/F12培养基再培养(对照组),然后进行如下实验:(1)分别于再培养24h、48h和72h,采用CCK-8检测两组HT29细胞增殖水平,并计算实验组HT29增殖率;(2)于再培养48h采用流式细胞术检测两组HT29细胞凋亡率;(3)再培养48h后,采用实时荧光定量PCR检测两组HT29细胞凋亡因子Caspase-3、Caspase-9、Survivin、XIAP的mRNA表达水平。结果:(1)与对照组比较,实验组HT29细胞24h增殖水平(吸光度OD值)无明显差异(P0.05),培养48h、72h的OD值显著降低(P0.01),且其增殖率下降趋势与培养时间呈明显负相关(r=-0.974,P0.01)。(2)实验组HT29细胞ASC-CM培养48h凋亡率较对照组明显增加(P0.01)。(3)实验组HT29细胞ASC-CM培养48h凋亡因子Caspase-3、Caspase-9mRNA表达较对照组显著升高(P0.01),Survivin、XIAP mRNA表达较对照组显著降低(P0.01)。结论:ASC-CM能有效抑制结肠癌HT29细胞增殖和促进凋亡。     

藤黄酸对人结肠癌HT-29细胞生物学行为及JNK信号通路的影响

目的探讨藤黄酸对人结肠癌HT-29细胞增殖、凋亡、迁移及ASK-1、p-ASK-1、JNK、p-JNK表达的影响。方法将人结肠癌HT-29细胞分为空白对照组与藤黄酸组(0.5、1.0、2.5、5.0μmol/ml)。不同浓度藤黄酸干预24、48、72h后,MTT方法检测藤黄酸对HT-29细胞增殖的抑制作用。2.5μmol/ml藤黄酸干预48 h后,流式细胞术检测HT-29细胞的凋亡率;划痕实验检测藤黄酸对HT-29细胞迁移能力的影响;Western blot实验检测藤黄酸对HT-29细胞JNK、p-JNK、ASK-1、p-ASK-1的表达。结果不同浓度藤黄酸均能显著抑制HT-29细胞增殖,且呈时间和浓度依赖性。2.5μmol/ml藤黄酸干预48 h后,HT-29细胞凋亡率升高,迁移能力降低(P0.01);JNK、p-JNK、ASK-1、p-ASK-1蛋白表达水平均显著升高(P0.01)。结论藤黄酸能够抑制人结肠癌HT-29细胞增殖与迁移,并促进凋亡,其作用机制可能与调控JNK信号通路相关。     

逆转录病毒介导的小鼠IL-23基因在小鼠结肠癌细胞中的表达及其抗肿瘤活性

目的：获得表达小鼠IL-23(mIL-23)基因的小鼠结肠癌细胞株。方法：应用逆转录病毒载体，将mIL-23基因导入小鼠结肠癌细胞株Colon26，经G418筛选后获得表达mIL-23的阳性细胞克隆(Colon26／IL-23)。用PCR和RT—PCR检测目的基因的表达，用ELISA法检测mIL-23的产生及mIL-23诱导的小鼠脾细胞IFN-γ的产生，用MTT比色法检测Colon26／IL-23细胞和Colon26细胞的体外增殖，将Colon26／IL-23细胞接种于BALB／c小鼠的右侧背部皮下，观察其致瘤性结果：建立了可表达mIL-23基因的小鼠结肠癌细胞株。分泌至培养上清中的IL-23，可诱导小鼠脾细胞产生IFN-γ在体外Colon26／IL-23细胞的生长与Colon26细胞无明显不同，但其在体内的致瘤性下降，具有抗瘤作用。结论：Colon6／IL-23细胞可分泌IL-23并证明其具有抗瘤活性。     

低表达miR-21对苦参碱诱导的肝癌HepG2细胞凋亡的影响

目的:探讨miR-21低表达能否增强苦参碱(matrine,MAT)对肝癌细胞的促凋亡作用。方法:实时荧光定量PCR(RT-qPCR)检测不同浓度MAT作用HepG2细胞后miR-21的表达情况。流式细胞术检测miR-21低表达对MAT诱导的细胞凋亡的影响;RT-qPCR和Western blot检测miR-21低表达对MAT诱导的细胞凋亡中Bc1-2和Bax mRNA和蛋白表达水平的影响。结果:miR-21表达量随MAT作用浓度的增加而增加;miR-21低表达可促进MAT诱导的细胞凋亡,并促进Bax mRNA和蛋白的表达(P0.05),而抑制Bcl-2 mRNA和蛋白的表达(P0.05)。结论:miR-21低表达可通过抑制Bcl-2和促进Bax的表达来增强MAT诱导的HepG2细胞凋亡。     

Caspase-9途径在丁酸钠诱导结肠癌细胞株HT-29凋亡中的作用

目的： 探讨caspase-9途径在丁酸钠(NaBt)诱导人结肠癌细胞株HT-29凋亡中的作用。方法： HT-29细胞体外培养至对数生长期,分别及联合给予5.0 mmol/L丁酸钠、20 μmol/L z-VAD-fmk、z-DEVD-fmk、z-IETD-fmk、z-LEHD-fmk处理24 h,并设空白对照。以Annexin V-FITC法联合PI染色,流式细胞术检测细胞凋亡,JC-1染色检测线粒体膜电位变化,caspase活性检测试剂盒检测caspase-3、caspase-8、caspase-9的活性。结果： (1)丁酸钠诱导的HT-29细胞凋亡［（35.40±0.70）%］可被z-VAD-fmk抑制［（1.33±0.59）%］,亦可被z-DEVD-fmk抑制［（1.40±0.52）%］,并可被z-LEHD-fmk抑制［（1.27±0.91）%］,均P<0.01;但是z-IETD-fmk不能够抑制该作用［（32.10±2.33）%］,P>0.05;(2)丁酸钠干预HT-29细胞后,线粒体膜电位降低(5.53±0.91),z-VAD-fmk、z-DEVD-fmk、及z-LEHD-fmk 能够阻断这种作用(9.80±1.15, 10.23±0.50, 10.33±1.02), P<0.05;而z-IETD-fmk未显示对该作用的改变（5.93±1.31）, P>0.05;(3)丁酸钠干预HT-29细胞后,caspase-3、caspase-9的活性增高2-3倍,caspase-8的活性无显著变化,P>0.05。结论： 丁酸钠主要是通过线粒体途径,激活caspase-9,启动细胞凋亡环节,从而激发下游的效应caspases,诱导HT-29细胞凋亡。     

23-羟基白桦酸诱导LoVo细胞凋亡与细胞内活性氧的关系

目的： 研究23-羟基白桦酸诱导人结肠癌细胞株LoVo细胞凋亡过程中活性氧（ROS）的变化及其作用机制。方法：光学显微镜法观察23-羟基白桦酸作用LoVo细胞后细胞形态学改变，并采用流式细胞仪检测细胞凋亡及细胞内ROS水平。结果：分别以浓度为25、50、100、200 μmol/L 23-羟基白桦酸作用LoVo细胞48h后，在光学显微镜下观察到明显的凋亡细胞形态学改变；经流式细胞仪检测，细胞凋亡率分别为(7.17±2.31)%、(15.60±4.02)%、(32.47±5.25)%及(52.71±5.93)%，呈现一定的浓度依赖性关系。23-羟基白桦酸可使 LoVo 细胞内ROS的水平明显高于空白对照组。细胞内ROS含量（MFI，平均荧光强度）分别为2.83±0.80、5.97±1.72、12.53±2.57及16.73±4.58。其中，浓度为100和200 μmol/L 23-羟基白桦酸的作用较明显，与对照组(2.13±0.32)比有显著差异（P<0.05）。结论：23-羟基白桦酸具有明显的诱导LoVo细胞凋亡作用，其作用机制可能与23-羟基白桦酸引起细胞内ROS水平增加有关。     

TRAIL联合氟尿嘧啶体外诱导结肠癌细胞凋亡

目的:探讨氟尿嘧啶(Fu)对重组人肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-relatedapoptosis-inducing ligand,TRAIL)诱导结肠癌细胞凋亡的影响及其作用机制。方法:采用四甲基偶氮唑蓝比色法检测Fu及联合rmhTRAIL对人结肠癌SW480的IC50值;流式细胞术检测不同浓度Fu与rmhTRAIL联合处理SW480 24 h后细胞的凋亡率;实时荧光定量PCR和Western印迹法检测不同浓度Fu与rmhTRAIL联合处理SW480 24 h后细胞survivin基因mRNA和蛋白表达变化。结果:细胞经药物处理24 h后,单用组IC50值为29.5μmol/L,Fu联合rmhTRAIL组IC50值为6.4μmol/L,高质量浓度联合组药物相互作用系数为0.92;Fu与rmhTRAIL联合作用24 h后,随Fu质量浓度增高survivin mRNA和蛋白的表达显著下调。结论:Fu能增强TRAIL诱导的结肠癌细胞凋亡,其机制可能与下调survivin表达有关。     

miR-23 regulates cell proliferation and apoptosis of vascular smooth muscle cells in coronary heart disease

BackgroundAberrant proliferation and migration of vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of cardiovascular diseases including coronary heart disease (CHD). MicroRNAs has reported play critical roles in VSMCs function. The present study was to investigate the effects of microRNA‑23 (miR-23) on VSMCs and uncover its potential mechanism.MethodsCell viability was detected by CCK-8 assay. Cell apoptosis was measured by flow cytometry. Dual luciferase reporter assay was conducted to verify whether BCL2L11 is a target gene of miR-23. The protein levels of BCL2L11 and caspase-3 were detect by quantitative real time PCR and western blot.ResultsOur results showed that the expression of miR-23 was upregulated in peripheral blood of CHD patients compared with controls. Overexpression of miR-23 promoted VSMCs proliferation and inhibited VSMCs apoptosis. Downregulation of miR-23 suppressed VSMCs proliferation and promoted VSMCs apoptosis. In addition, we identified BCL2L11 was a direct gene of miR-23. Overexpression of miR-23 decreased the levels of BCL2L11 and caspase-3, and downregulate of miR-23 increased the levels of BCL2L11and caspase-3 in VSMCs.ConclusionOur findings suggest that miR-23 plays a crucial role in controlling VSMCs proliferation and apoptosis by targeting BCL2L11.     

Induction of apoptosis in HT-29 cells infected with SA-11 rotavirus

Rotavirus infection is associated both in vivo and in vitro with a series of subcellular pathological alterations leading to cell lysis. It has been suggested that these modifications can play a key role in the pathogenesis of rotavirus-associated diarrheal disease. We describe the effects of SA-11 rotavirus infection in HT-29 cells, a human enterocyte-like cell line. Cytological analyses suggested that the viral-induced cytopathic process, including chromatin clumping, can be referred to as apoptosis, the cell death pathway alternative to necrosis. A time course of the process was performed to investigate whether rotavirus-associated cell death showed specific injury signs. HT-29-infected cells were analyzed by scanning and transmission electron microscopy and features of apoptosis such as blebbing of the plasma membrane, peripheral condensation of chromatin, and fragmentation of the nucleus were observed. Specific changes occurring in cell-substrate adhesion and in some organelles relevant for viral maturation, i.e., rough endoplasmic reticulum, were detected. These findings indicate a role for apoptosis in the rotavirus infection process and its related cytopathology, and also suggested that specific histological alterations such as derangement of enterocytes are associated with the pathogenesis of rotavirus-induced diarrheal disease and could be a direct consequence of viral-triggered apoptosis. © 1996 Wiley-Liss, Inc.     

miR-23a和ESRP1在直肠癌中的作用

目的:研究miR-23a和上皮剪接调节蛋白1(epithelial splicing regulatory protein 1,ESRP1)在直肠癌组织及细胞系中的表达,以及对体外直肠癌细胞活力和凋亡的作用。方法:采用RT-q PCR分析miR-23a在36例直肠癌组织和癌旁组织中的表达,免疫组化检测ESRP1在直肠癌组织中的表达,分析miR-23a和ESRPl在直肠癌组织中的相关性;利用RT-q PCR检测miR-23a在直肠癌Caco-2和SW480细胞及人正常结肠上皮细胞株NCM460中的表达;合成miR-23a inhibitor和inhibitor阴性对照(inhibitor NC),并将其分别转染至SW480细胞后,通过CCK-8法检测miR-23a inhibitor转染SW480细胞后对细胞活力的影响,流式细胞术检测转染后细胞凋亡率,Transwell小室实验检测细胞侵袭;通过Western blot技术检测SW480细胞中ESRPl蛋白的表达;构建野生型pGL3-ESRP1-3’UTR(wt-pGL3-ESRP1-3’UTR)或突变型pGL3-ESRP1-3’UTR(mut-pGL3-ESRP1-3’UTR)质粒,并分别与miR-23a inhibitor或inhibitor NC共转染至HEK293和SW480细胞中,利用双萤光素酶报告基因检测试剂盒说明检测双萤光素酶活性;将ESRP1 mimic或mimic NC瞬时转染SW480细胞后,CCK-8法和流式细胞术分别检测细胞活力和凋亡;Western blot法检测瞬转ESRP1 mimic后对ESRP1、caspase-3、Smac和XIAP蛋白表达的影响。结果:miR-23a和ESRP1在直肠癌组织的表达较癌旁正常组织分别上调和下调,两者呈明显负相关(P0.01);miR-23a的表达与直肠癌的淋巴结转移和肿瘤浸润深度相关;与NCM460细胞相比较,miR-23a在SW480细胞中的表达量显著上调(P0.01);转染miR-23a inhibitor后,SW480细胞活力较inhibitor NC组显著下降(P0.01);转染miR-23a inhibitor后SW480细胞早期凋亡率明显升高,同时细胞体外侵袭能力受到抑制;萤光素酶报告基因结果表明ESRP1是miR-23a的直接靶基因;转染miR-23a inhibitor至SW480细胞后ESRP1蛋白表达水平明显升高;ESRP1 mimic转染SW480细胞后可抑制细胞活力并诱导细胞凋亡,同时上调caspase-3和Smac的表达,下调XIAP的表达。结论:miR-23a可通过负向调控下游靶基因ESRP1从而影响直肠癌细胞生长和凋亡。     

Effect of indomethacin on E-cadherin and beta-catenin expression in HT-29 colon cancer cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) lower the incidence of and mortality from colon cancer. Although there is much evidence from epidemiological and laboratory studies that NSAIDs have antitumor activity and reduce the incidence of colon cancer, the mechanism of action remains unknown. In this paper, we present the effect of indomethacin on growth inhibition, induction of apoptosis, and alterations in the expression of several genes involved in Wnt signaling in HT-29 colon cancer cells. We have shown that indomethacin reduces the proliferation rate of HT-29 colon cancer cells and induces apoptosis. Concentrations of indomethacin from 10(-4) to 10(-3) M strongly inhibited the growth of HT-29 cells. The inhibition of growth, as well as induction of apoptosis was dose and time dependent. The treatment of cells with 4 x 10(-4) M indomethacin caused strong inhibition of cell growth (about 70%), enhanced expression of APC, decreased expression of beta-catenin and induced expression of E-cadherin proteins. Expression of beta-catenin was not markedly reduced instead, beta-catenin was translocated from the nucleus and cytoplasm to the plasma membrane. These results were confirmed by real-time RT-PCR analysis on mRNA level. At a concentration of 4 x 10(-4) M indomethacin there was increased expression of APC gene (10.9-fold induction; DeltaDeltaCt = 3.43) and E-cadherin gene (3.5-fold induction; DeltaDeltaCt = 1.79). These results suggest the antiproliferative effect of indomethacin may contribute to enhanced cell adhesion through increased expression of E-cadherin and translocation of beta-catenin from the nucleus to the cell membrane.     

Aldolase B impairs DNA mismatch repair and induces apoptosis in colon adenocarcinoma

Evidence suggests that DNA repair capacity manifested by intact functional base excision repair and mismatch repair (MMR) pathways is related to the prognosis of multiple cancer types. Aldolase B (ALDOB) is well known for its role in metabolism and glycolysis. The expression of ALDOB in colon adenocarcinoma and the relationship between its expression and colon adenocarcinoma prognosis remain controversial; in addition, the potential role of ALDOB in DNA MMR has not yet been reported. In this study, we identified a cluster of DNA repair-related proteins that interact with ALDOB in the colon adenocarcinoma cell line HCT116. Expression analysis of colon adenocarcinoma data from the Cancer Genome Atlas (TCGA-COAD data, n = 551) indicated that ALDOB mRNA expression was significantly higher in specimens with microsatellite instability (MSI) than in specimens with microsatellite stability (MSS). Regarding prognosis, colon adenocarcinoma patients with high ALDOB mRNA expression had longer overall survival (OS). Higher expression of ALDOB protein was significantly correlated with MMR deficiency (d-MMR) in formalin-fixed paraffin-embedded (FFPE) patient specimens. The expression of ALDOB was significantly elevated in colon adenocarcinoma cell lines. Further evidence indicated that rather than affecting proliferation, ALDOB overexpression induced the functional loss of MMR proteins and in turn caused irreversible DNA damage via disrupting EZH2-Rad51 expression and then caused apoptosis by ERK inactivation. Overall, our study demonstrates that high ALDOB expression impairs DNA MMR and induces apoptosis in colon adenocarcinoma. ALDOB may be a new biomarker associated with d-MMR and an independent prognostic factor for colon adenocarcinoma.     

三氧化二砷诱导卵巢癌细胞凋亡的研究

目的在体外实验中,探讨三氧化二砷(As2O3)诱导卵巢癌细胞发生凋亡的可能性,揭示该凋亡发生与bcl-2和bax之间的关系。方法采用TUNEL染色法,定性、定量地研究As2O3与卵巢癌细胞凋亡的关系;通过免疫组织化学法检测凋亡相关基因bcl-2和bax的表达。结果As2O3在体外能诱导卵巢癌细胞发生凋亡,下调bcl-2的表达,增强bax的表达。结论诱导卵巢癌细胞发生凋亡是As2O3抗卵巢癌作用的机制之一,As2O3可能通过下调bcl-2的表达及增强bax的表达诱导卵巢癌细胞发生凋亡。