丁酸钠诱导MCF-7细胞凋亡过程中端粒酶活性的变化

目的　研究丁酸钠诱导MCF 7细胞凋亡过程中端粒酶活性变化及其机制。方法　采用MTT法和倒置相差显微镜观察不同浓度丁酸钠对MCF 7细胞的抑制作用 ,流式细胞仪和琼脂糖凝胶电泳检测 2 .5mmol/L丁酸钠作用后的细胞凋亡情况 ,TRAP ELISA法检测端粒酶活性变化 ,RT PCR分析端粒酶各组分的mRNA表达情况。结果　丁酸钠对MCF 7细胞的抑制作用具有时间和剂量依赖性 ,AnnexinV/PI双染法显示 ,2 .5mmol/L丁酸钠作用 72h后 ,细胞凋亡率为 84 .3% ,琼脂糖凝胶电泳可见间隔为 180bp的DNA梯状条带。 2 .5mmol/L丁酸钠作用 2 4和 4 8h后 ,端粒酶活性分别下降为1.82± 0 .2 2和 1.6 1± 0 .0 9。RT PCR显示 ,端粒酶逆转录酶 (hTERT)表达下降 ,而端粒酶RNA模板(hTR)和端粒酶相关蛋白 (hTP1)表达无明显改变。结论　丁酸钠诱导MCF 7细胞凋亡过程中端粒酶活性下降 ,其机制可能与丁酸钠下调hTERT转录水平有关。     

建立胶质瘤细胞诱导分化相关基因谱

目的 建立胶质瘤诱导分化相关基因表达谱。方法 用苯丁酸钠诱导胶质瘤细胞分化,cDNA阵列检测诱导前和诱导2h、6d后的18000个基因表达,扫描仪分析结果,多点杂交验证。结果 在胶质瘤细胞诱导分化后,有98个基因发生表达变化,一些转录和翻译系统相关基因和癌基因表达下调,而分化、凋亡基因上调。还有18个未知基因EST片段分化前后表达差异。结论 含有98个基因的胶质瘤细胞诱导分化相关基因 可为探讨其分化分子机制提供依据。     

松胞素 B对白血病 K562细胞基因表达谱的影响

背景与目的：近年来兴起的DNA芯片技术能在一次杂交中同时监测数以千计的基因表达,能大大加速肿瘤药作用机制的研究和药物治疗靶点的发现。本研究的目的是利用基因表达谱芯片研究松胞素B（cytochalasinB,CB,旧称细胞松弛素B）对白血病K562细胞基因表达谱的影响。方法：利用限制性显示RCR（restriction disphalPCR,RD-PCR)技术扩增K562细胞cDNA片段。取其中277个cDNA片段按设计的矩阵打印在玻片上,制成基因芯片;用CB处理K562细胞24h,分别提取对照组和处理组细胞总RNA,将两组等量的细胞总RNA纯化为mRNA,反转录为cDNA,利用RD-PCR技术进行扩增标记;与芯片杂交后扫描分析处理前后表达差异的基因。结果：在所收集的探针中,对照组和处理组细胞间存在差异表达的基因。共筛选到CB处理后表达下调的基因18条。结论：CB诱导后的K562细胞部分基因表达呈下调趋势,其中大部分基因与细胞增殖,信号转导,转录调节相关。     

沙棘籽渣黄酮诱导人乳腺癌细胞凋亡相关基因的表达谱变化

背景与目的:研究显示,沙棘含有多种黄酮类化合物,具有抗氧化、防辐射等作用。cDNA基因表达谱芯片技术具有高通量、高效、快捷的特点,本实验拟利用cDNA基因表达谱芯片研究沙棘籽渣黄酮(flavonoidsfromseedresiduesofHippophaerhamnoidesL.,FHR)诱导人乳腺癌细胞Bcap鄄37凋亡相关基因的表达谱变化。方法:抽提FHR作用前后的Bcap鄄37细胞总RNA,经逆转录合成荧光分子(Cy3/Cy5)标记的cDNA探针,与含有13824个cDNA基因的人14K基因表达谱芯片杂交,利用Genespring软件分析实验组和对照组之间差异表达的基因,对所获得的基因进行生物信息学分析。结果:实验组与对照组中表达上调的基因有305条,表达下调的基因有361条。差异表达的与细胞凋亡相关的基因有32条,占芯片基因总数的0.23%,其中表达上调的有25条(平均Ratio值为3.071),下调的有7条(平均Ratio值为0.418)。生物信息学分析表明,FHR在对Bcap鄄37细胞作用的过程中,该32条差异表达基因与Bcap鄄37细胞凋亡有关,其中包括CTNNB1、TSSC3、IGFBP4、IGFBP6、GADD34、TNFRSF10B、Caspase鄄9和PCNA等。结论:FHR诱导Bcap鄄37细胞凋亡的机制是由多基因协同,并通过胞内和胞外信号转导途径共同调节完成。     

苯丁酸钠体外对人肝癌细胞HepG2增殖和凋亡的影响

[目的]探讨组蛋白去乙酰化酶抑制剂苯丁酸钠（PB）体外对人肝癌细胞HepG2增殖和凋亡的影响。[方法]不同浓度苯丁酸钠处理HepG2,应用MTT比色法观察苯丁酸钠对HepG2的生长抑制作用,落射荧光显微镜、DNA电泳和流式细胞仪观察HepG2的凋亡,West-ernblot检测苯丁酸钠处理前后HepG2细胞中Bcl-2、Bax蛋白表达水平的变化。[结果]苯丁酸钠2mmol/L、4mmol/L、8mmol/L作用48h对细胞的抑制率分别为19.41%、39.03%、42.19%,作用72h对细胞的抑制率分别为27.42%、57.11%、70.31%,并诱导细胞凋亡;4mmol/L苯丁酸钠作用HepG248h细胞凋亡率明显高于对照组。落射荧光显微镜和DNA电泳均观察到苯丁酸钠作用后HepG2细胞出现凋亡细胞的特征性变化。苯丁酸钠处理HepG2后Bcl-2蛋白表达减少,Bax蛋白表达增加。[结论]苯丁酸钠体外抑制HepG2增殖并促进其凋亡,其作用机制可能是通过降低细胞内的Bcl-2蛋白,增加细胞内Bax蛋白而实现的。     

丁酸钠诱导HT 29结肠癌细胞凋亡及其对 p 53靶基因的调控

目的：〖HT5"SS〗分析丁酸钠对HT29结肠癌细胞p53三个主要靶基因（p21waf1,bax和gadd45）的调控,并探讨其作用机制。〖HT5W〗方法：〖HT5"SS〗HT29细胞常规培养在含有和不含有丁酸钠的培养液中。分别用MTT和流式细胞仪(flow cytometry,FCM) 检测细胞增殖和细胞周期分布,通过形态学观察、亚G1峰的检测和AnnexinVFITC 双标记观察细胞凋亡情况;RTPCR和Western blot分别检测丁酸钠对p21waf1,bax和gadd45三种基因mRNA和蛋白表达水平的影响。〖HT5W〗结果：〖HT5”SS〗丁酸钠以剂量和时间依赖的方式抑制HT29细胞增殖和诱导凋亡,并阻滞细胞于G1期。RTPCR 和Western blot结果显示丁酸钠可以促进p21waf1和bax基因mRNA和蛋白的表达,而对gadd45基因的表达无明显影响。〖HT5W〗结论：〖HT5”SS〗2.5 mmol/L以上浓度的丁酸钠可以抑制HT29细胞增殖并诱导凋亡,该作用可能通过上调p21waf1和bax基因表达而实现。     

冬凌草甲素对人类食管癌SHEEC细胞基因表达的影响及其靶基因筛选

 目的　检测冬凌草甲素（ORI）对人类食管癌SHEEC细胞基因表达的影响并筛选与细胞凋亡密切相关的靶基因。方法　应用基因芯片技术检测32 μg／ml ORI作用SHEEC细胞1 h及8 h前后的基因表达差异,分析筛选差异表达基因,并用荧光定量PCR进行验证。结果　32 μg／ml ORI作用SHEEC细胞1 h及8 h后,表达上调≥2倍及表达下调≥2倍的基因共1011个,其中,1 h有 280个,8 h有731个。在1011个差异表达基因中,最后筛选出17个重复检测表达上调或表达下调幅度大且与细胞凋亡、信号转导和转录调控相关的基因。荧光定量PCR对17个差异表达基因进行验证,其中12个基因具有统计学意义。结论　在12个有统计学意义的差异表达基因中,可能有ORI经线粒体途径诱导肿瘤细胞凋亡的靶基因。     

吉西他滨诱导人NK/T细胞淋巴瘤细胞株SNK-6凋亡的机制

目的探讨吉西他滨诱导人NK/T细胞淋巴瘤细胞株SNK-6产生凋亡的机制。方法应用MTT法、DNA电泳技术及流式细胞术观察吉西他滨诱导人SNK-6细胞凋亡情况,应用基因芯片技术分析加药前后凋亡相关基因的表达差异。结果吉西他滨作用后,MTT结果显示细胞生长抑制率随药物浓度的增加而逐渐增大;DNA断裂电泳可见典型的梯形DNA条带,随吉西他滨作用浓度的增加而逐渐增强;流式细胞术分析,2 μg/ml吉西他滨作用SNK-6细胞4、8、24、48 h后,凋亡细胞比例分别为2.1%、8.3%、23.9%、30.9%;吉西他滨作用48 h后,在88条有关凋亡的目的基因中共有17条基因表达发生大于3倍的显著变化,其中表达上调基因4条,下调基因13条。结论吉西他滨可以诱导人NK/T细胞淋巴瘤细胞产生凋亡,呈时间和剂量依赖性;吉西他滨可使SNK-6细胞中多个凋亡相关基因的表达发生改变,初步揭示了其诱导淋巴瘤细胞凋亡的作用机制。     

丁酸钠对人胃癌细胞的增殖抑制和凋亡诱导作用机制研究

目的：研究丁酸钠对人胃癌细胞系生长的抑制和诱导凋亡作用,探讨其作用机制。方法：以不同浓度的丁酸钠对体外培养的人胃癌细胞系SGC-7901进行处理,应用MTT法检测对细胞生长的抑制情况;应用免疫组化法观察对凋亡相关基因p21WAF1表达的影响。结果：不同浓度的丁酸钠均可抑制SGC-7901细胞的增殖,且抑制率具有剂量和时间依赖性;p21WAF1在丁酸钠作用的细胞中的表达高于未进行处理的细胞。结论：丁酸钠可抑制体外培养的人胃癌细胞生长,诱导癌细胞发生凋亡。     

BCL—2蛋白不能抑制VP16诱导的乳腺癌细胞（Bcap37）凋亡

目的 为了解BCL—2在Bcap37细胞表达增高能否抑制足叶乙甙诱导的细胞凋亡,把重组的逆转录表达载体pLXSN—bcl—2和pLXSN—neo转染Bcap37细胞,经G418筛选获得表达细胞(Bcap37—bcl—2,Bcap37—neo)。方法 经RT—PCR和Western blotting检测bcl—2基因的表达,并用不同浓度的VP16处理两种细胞16小时,经细胞形态观察、细胞凋亡原位显示、MTT实验检测细胞的凋亡变化。结果 两种细胞的凋亡变化没有显著差异。结论 BCL—2高表达不能阻滞VP16诱导的Bcap37细胞凋亡,这与我们以前所做的关于BCL—2能阻滞VP16诱导的K562细胞凋亡作用相反,推测BCL—2的抗凋亡作用可能和细胞类型有关,可能还有其它因素参与BCL—2对细胞凋亡的调节。     

Sodium butyrate-induced DAPK-mediated apoptosis in human gastric cancer cells

Epigenetic mechanisms of histone acetylation/deacetylation play an important role in the regulation of gene expression associated with the cell cycle and apoptosis. Recently, sodium butyrate, a histone deacetylase (HDAC) inhibitor, has been shown to exhibit anticancer effects via differentiation and apoptosis of cancer cells. Sodium butyrate may be a potential anticancer chemotherapeutic drug; however, the precise mechanism underlying the anticancer effects of sodium butyrate has not been clearly elucidated. In the present study, we investigated the role of death-associated protein kinase (DAPK) on the apoptosis of human gastric cancer cells induced by sodium butyrate. We observed that sodium butyrate induced apoptosis in human gastric cancer cells. Treatment with the HDAC inhibitor sodium butyrate increased the expression of caspase-3 and DAPK1/2 genes but decreased the expression of Bcl-2 in human gastric cancer cells. The expression of DAPK3, p53 and p21 were not altered by sodium butyrate treatment. Analysis of the general expression patterns revealed that sodium butyrate increased the expression of DAPK1/2 but decreased the expression of FAK and induced changes in the proliferation of apoptosis-related genes in human gastric cancer cells. These data suggest that DAPK expression prompts apoptosis by reducing the FAK protein level in sodium butyrate-induced apoptosis of human gastric cancer cells.     

Gene expression associated with the decrease in malignant phenotype of human liver cancer cells following stimulation with a histone deacetylase inhibitor

Sodium butyrate is a short-chain fatty acid produced by fermentation in the gastrointestinal tract. It induces differentiation of several kinds of cancer by inhibiting histone deacetylase activity. We have reported that butyrate stimulates hepatocellular carcinoma cells into their normal phenotype. Since sodium butyrate affects both differentiation and apoptosis, we investigated expression of bcl-2-related genes in a human hepatocellular carcinoma cell line HCC-T. The expression of anti-apoptotic Bcl-2 and Mcl-1/EAT was up-regulated 4 h after the treatment, while pro-apoptotic Bax expression did not change. Gene expressions in the early stage of butyrate-stimulation were investigated by the differential display assay and the cDNA expression array. Laminin and keratin 18 were increased 6 h after the stimulation with sodium butyrate. The results of cDNA expression array revealed up-regulation of cell cycle inhibitory genes such as cyclin-dependent kinase 4 inhibitor, and interferon-related genes such as STAT2 and 3, while down-regulation of cyclin-dependent kinase 2 and cyclin E. Up-regulated production of p21WAF-1 and Mcl-1/EAT was also confirmed by Western blotting. The cytoskeletal change indicated by up-regulation of laminin and keratin 18 may be an important factor in the decrease in malignant phenotype of cancer cells. Up-regulation of interferon-related genes indicated that butyrate-treatment might induce a similar phenotypic change to that induced by type 1 interferons. This study suggests several target genes for the future gene therapy of cancer or genes preventing cancer development from pre-malignant tissues.     

Apoptosis induced by the sodium butyrate in human gastric cancer TMK-1 cells

The effects of sodium butyrate on cell proliferation, gene expression, and apoptosis were investigated. Upon exposure to sodium butyrate the cells exhibited marked morphological changes, reduced cell proliferation and most cells died through apoptosis within 48 hours. In the presence of dexamethasone, however, the sodium butyrate-triggered apoptosis was markedly reduced. Studies using the glucocorticoid receptor antagonist RU486 indicated that the protective effect of dexamethasone was mediated through glucocorticoid receptor. Sodium butyrate markedly induced the c-jun proteins level, whereas the c-Myc protein was down-regulated rapidly. c-Jun protein may play an important role in the action of sodium butyrate since its induction preceded the onset of DNA fragmentation. In addition, preincubation of the cells with dexamethasone markedly delayed the induction of c-jun levels by sodium butyrate. Analysis of the expression of bel-2-related genes indicated that the Bcl-xS protein level was increased in the presence of sodium butyrate and the up-regulation of Bcl-xS by sodium butyrate was also blocked by dexamethasone. Taken together, these results indicate that c-myc, c-jun and Bcl-xS proteins may be involved in the mechanism of sodium butyrate-triggered apoptosis in these cells.     

丁酸钠对前列腺癌LNCaP细胞HER-2信号通路的影响

 目的 研究组蛋白去乙酰化酶抑制剂丁酸钠对前列腺癌LNCaP细胞HER 2信号通路的影响，探讨其抗肿瘤作用的分子机制。方法 四甲基偶氮唑蓝(MTT)检测药物对肿瘤细胞增殖的影响；hoechst 33342染色观察细胞凋亡的形态学变化，Western blot检测凋亡标志蛋白、HER2/ neu、Phos Akt、Phos Erk等信号蛋白的表达。结果 丁酸钠能够有效抑制LNCaP细胞的增殖并诱导细胞凋亡，半效杀伤剂量（EC50）为5.6mmol/L；药物能够抑制HER 2基因的转录和蛋白的表达，并抑制下游信号通路中MAPK和AKT的活化。结论 丁酸钠能够阻断对前列腺癌细胞生长具有重要作用的HER 2信号通路，从而对肿瘤细胞发挥抑制作用。     

丁酸钠诱导MCF-7细胞凋亡的研究

目的:观察丁酸钠诱导培养的乳腺癌细胞MCF-7凋亡的影响。方法:用终浓度分别为0、1.25、2.5、5.0、10.0mmol/L的丁酸钠处理MCF-7细胞48h,用MTT比色法分析丁酸钠对细胞的抑制率,倒置显微镜观察细胞生长情况的改变,流式细胞术,末端脱氧核糖核酸转移酶介导的缺口末端标记法(TUNEL)及DNA琼脂糖凝胶电泳检测凋亡。结果:不同浓度的丁酸钠对MCF-7细胞有明显的抑制作用,光镜下观察到细胞贴壁性随作用时间延长而下降;流式细胞术检测不同浓度丁酸钠处理细胞的凋亡率分别为4.8%、9.4%、26.1%和51.9%;TUNEL检测可见阳性细胞胞体缩小,核固缩,呈棕黄色或黄褐色颗粒;DNA琼脂糖凝胶电泳检测到10mmol/L处理细胞出现“DNA ladders”。结论:丁酸钠可以诱导MCF-7细胞发生凋亡。     

bcl-2 and bak may play a pivotal role in sodium butyrate-induced apoptosis in colonic epithelial cells; however overexpression of bcl-2 does not protect against bak-mediated apoptosis

Butyrate, a short chain fatty acid produced in the colon as a result of fermentation of dietary fibre by symbiotic bacteria, induces apoptosis in colonic tumour cell lines. Three human colonic adenoma cell lines (AA/C1, RG/C2 and BH/C1) and one carcinoma cell line (S/KS/FI) were used to determine the effects of butyrate on the expression of bcl-2, bax and bak to examine the possible role of these proteins in the induction of apoptosis. RG/C2 and BH/C1 cells express p-26-bcl-2 and butyrate treatment decreased p26-bcl-2 levels in association with apoptosis, whereas bax and bak levels remained constant. AA/C1 and S/KS/FI cells have no detectable p26-bcl-2. In S/KS/FI cells, bax or bak levels did not change in response to butyrate. However, in AA/C1 cells, butyrate-induced apoptosis was associated with increased bak levels. Therefore, in AA/C1 cells butyrate-induced apoptosis appears to be mediated through bak. Furthermore, butyrate also induced apoptosis and increased bak levels in AA/C1 cells transfected with a bcl-2 expression vector which expressed high levels of p26-bcl-2. For S/KS/FI cells, two bcl-2 transfectants gave different results. bcl-2 protected against apoptosis in one transfectant in which bak levels were not elevated in response to butyrate, whereas it did not protect in the other transfectant in which bak levels were increased after butyrate treatment. The results suggest that expression of constitutively high levels of p26-bcl-2 only conferred protection against apoptosis when bak levels were not elevated in response to butyrate and that expression of constitutively high levels of p26-bcl-2 does not counter the effects of bak. Different mechanisms appear to be involved in cell death signalling in different tumours since butyrate may induce apoptosis via elevated levels of bak or reduced levels of p26-bcl-2. Int. J. Cancer 72:898–905, 1997. © 1997 Wiley-Liss, Inc.