新生牛牛脑活性肽NBBP-1对人神经母细胞瘤SK-N-SH细胞增殖和相关基因表达的影响

目的:应用新生牛牛脑活性肽NBBP-1处理人神经母细胞瘤SK-N-SH细胞,观察NBBP-1对人神经母细胞瘤SK-N-SH细胞增殖和相关基因表达的影响.方法:通过细胞计数、流式细胞仪、光学显微镜、免疫细胞化学方法检测细胞的变化.结果:经60μg/mL NBBP-1处理后,SK-N-SH细胞生长抑制率高达90.09%,细胞周期被阻滞在G0/G1,细胞核裂解成多个,免疫细胞化学染色结果显示,经处理后bcl-2抗凋亡基因蛋白表达减弱,而p53、fas、bax等促凋亡基因蛋白表达增强.结论:新生牛牛脑活性肽NBBP-1对人神经母细胞瘤SK-N-SH细胞凋亡具有显著的诱导作用,其诱导癌细胞凋亡的机理与其调节和干预癌基因bcl-2和p53、fas、bax等抑癌基因的表达有关.     

PUMA在结肠癌细胞中的表达及诱导细胞凋亡中的作用

目的:通过5-Fu诱导携带有野生型p53基因的HCT116和携带有突变性p53基因的HT-29两种结肠癌细胞系,比较两株细胞在各时间点凋亡水平和PUMA mRNA表达情况的差异,探讨PUMA对细胞凋亡的作用及诱导其表达的基本途径。方法:用逆转录聚合酶链反应(RT—PCR)检测不同结肠癌细胞株HT-29、HCT116在抗肿瘤药物5-Fu作用下不同时间点结肠癌细胞株内PUMA mRNA表达水平的差异,用吖啶橙/溴化乙啶(AO/EB)荧光染色检测各时间点细胞的凋亡水平,分析其与PUMAmRNA表达水平之间的关系。结果:携带有野生型P53基因的结肠癌细胞株HCT116在5-Fu作用下6h即可出现PUMA mRNA的表达,随着药物作用时间的延长其表达强度增加,并且与细胞凋亡水平呈正相关;含有突变型P53基因的结肠癌细胞株HT-29在5-Fu作用下无PUMA的表达。结论:通过5-Fu诱导细胞凋亡出现的PUMA表达需要野生型P53基因,突变型P53基因无法诱导PUMA的表达。PUMA与结肠癌细胞凋亡水平呈正相关,是一种促凋亡蛋白。     

p53、bax和bcl-2基因在SO 2染毒大鼠肝中的表达

为了进一步探讨SO₂的毒理学作用机制,运用荧光实时定量RT-PCR和免疫组化技术研究SO2吸入对大鼠肝细胞中p53、bax和bcl-2三种细胞凋亡相关基因mRNA和蛋白表达的影响.结果显示,肝中p53和bax mRNA水平呈剂量依赖性增加,在SO ₂浓度为28.00和56.00 mg/m3时显著增加（p53 mRNA在28 mg/m ³为1.30倍,在56 mg/m^3为3.43倍;bax mRNA在28 mg/m3为1.63倍,在56 mg/m ³为2.17倍）;而bcl-2 mRNA水平显著降低（在28 mg/m3为0.63倍,在56 mg/m ³为0.45倍）.免疫组化实验结果表明,吸入SO ₂后,大鼠肝中p53和bax蛋白表达水平呈剂量依赖性增加,而bcl-2蛋白表达水平呈剂量依赖性降低.结果表明,SO2可以改变凋亡相关基因的表达,诱导大鼠肝组织细胞凋亡,这可能与一些凋亡相关疾病的发生有关.     

刺五加多糖诱导人小细胞肺癌H446 细胞凋亡

研究刺五加多糖(Acanthopanax senticosus polysaccharide,ASPS)诱导H446细胞凋亡及其可能的作用机制.采用MTT法检测ASPS对小细胞肺癌H446细胞增殖的抑制作用;Hoechst 33258染色和流式细胞技术检测经ASPS处理后H446细胞凋亡的形态特征及凋亡率的变化;West ern印迹方法检测凋亡相关基因bax、bcl-2、p53 表达的变化.MTT分析表明,ASPS作用48 h后可明显抑制H446细胞的增殖,半数抑制浓度(IC50值)为476.36 mg/ml;Hoechst 染色结果: H446细胞在ASPS诱导下出现典型的凋亡形态;流式细胞术检测结果显示: 对照组及浓度为240、480、960 mg/ml 药物处理组凋亡率分别是(5.02±0.4)%、(11.12±0.8)%、(19.89±0.5)%、(22.54±0.8)%;Western印迹显示: 在ASPS的诱导下bax、p53的表达量提高,而bcl-2的表达量下降.研究表明,ASPS对H446细胞增殖有抑制作用,并能促进其凋亡;ASPS通过上调bax、 p53表达,下调bcl-2表达促进H446细胞凋亡.     

猴免疫缺陷病毒引起多形核嗜中性白细胞凋亡的可能机理

本实验目的是研究猴免疫缺陷病毒(SIV)引起多形核嗜中性白细胞(PMNs)凋亡的机理.实验用PCR技术扩增gag基因,用Western blot法测定p53和bcl-2基因的表达.结果显示PMNs在被SIV感染后随着保温时间的延长存活率下降,在感染后24h可以从PMNs中扩增出gag基因.PMNs中p53基因的的表达在感染后24h增加.同时bcl-2基因的表达在对照组和SIV感染组都增加,但在SIV感染组bcl-2蛋白的表达明显低于对照组.结果揭示SⅣ能够感染PMNs,p53和bcl-2基因表达的改变可能是SⅣ感染PMNs引起细胞凋亡的机理.     

bc1-2和bax及NF-kB在白藜芦醇诱导肝癌细胞凋亡中的作用

目的探讨白藜芦醇诱导肝癌细胞凋亡的途径.方法白藜芦醇体外处理HepG2肝癌细胞24 h后,以免疫组化检测凋亡调控基因bcl-2和bax及NF-kB的表达.结果白藜芦醇处理组HepG2细胞bcl-2的阳性积分和NF-kB的阳性细胞密度均明显低于对照组(P＜0.01);而bax阳性积分明显高于对照组(P＜0.01).结论白藜芦醇能下调HepG2细胞bcl-2基因的表达,上调bax的表达,同时抑制NF-kB的活化,这可能是其诱导HepG2细胞凋亡的途径之一.     

重组腺病毒介导人野生型p53和B7-1基因诱导喉癌细胞凋亡并增强其免疫原性

为开展喉癌的复合基因治疗,观察共表达人野生型p53和B7-1基因的重组腺病毒Ad-p53/B7-1对喉癌细胞增殖及免疫原性的影响,检测到Ad-p53/B7-1介导的人野生型p53基因可抑制喉癌细胞增殖并诱导其凋亡,同时B7-1基因的导入可增强喉癌细胞的免疫原性基因修饰的癌细胞可刺激自体肿瘤浸润淋巴细胞增殖,并诱导外周血淋巴细胞形成对肿瘤具有特异杀伤性的细胞毒性T淋巴细胞。     

猴免疫缺陷病毒引起多形核嗜中性白细胞凋亡的可能机理（英文）

本实验目的是研究猴免疫缺陷病毒(SIV)引起多形核嗜中性白细胞(PMNs)凋亡的机理.实验用PCR技术扩增gag基因,用Western blot法测定p53和bcl-2基因的表达.结果显示PMNs在被SIV感染后随着保温时间的延长存活率下降,在感染后24h可以从PMNs中扩增出gag基因.PMNs中p53基因的的表达在感染后24h增加.同时bcl-2基因的表达在对照组和SIV感染组都增加,但在SIV感染组bcl-2蛋白的表达明显低于对照组.结果揭示SⅣ能够感染PMNs,p53和bcl-2基因表达的改变可能是SⅣ感染PMNs引起细胞凋亡的机理.     

PTEN基因诱导人胚肾293细胞凋亡和细胞周期停滞

为了研究抑癌基因PTEN过表达对HEK293细胞凋亡和细胞周期停滞的作用,以野生型PTEN和PTEN突变子(T910G)表达质粒分别转染无PTEN表达的人胚肾293细胞,采用细胞质梯度DNA方法检测细胞凋亡,以流式细胞仪分析细胞周期.发现PTEN过表达能够诱导人胚肾293细胞质中出现梯度DNA,293细胞发生凋亡,PTEN过表达改变细胞周期分布,G0/G1期细胞增加13%,S期细胞下降15%.PTEN突变子对细胞凋亡和G1细胞停滞的影响略弱于野生型PTEN.PTEN基因过表达明显下调血小板衍生生长因子(PDGF)诱导的蛋白激酶B(PKB)和p42,p44-促分裂原活化蛋白激酶(MAPK)磷酸化水平,PTEN突变子对p42,p44-MAPK磷酸化水平的调节作用略弱于野生型PTEN.PTEN通过抑制细胞增殖,诱导细胞凋亡而影响细胞生长.     

灵芝肽诱导人肝癌HepG2细胞凋亡的研究

研究了灵芝肽(GLP)在体外对人肝癌HepG2细胞凋亡的影响,并初步探讨了其作用机制。结果显示,透射电镜下可见细胞染色质浓缩、聚集于核边缘成块状,形成典型的凋亡小体;GLP使HepG2细胞阻滞于G0/G1期,随着GLP浓度升高,其G0/G1期的细胞比例随之增加;同时细胞的早期、晚期和总的凋亡率亦均随之增加,存在剂量-效应关系;Western blotting检测结果显示,抑制凋亡基因bcl-2和survivin表达下调,而促凋亡基因p53表达上调,并且都存在剂量依赖性;细胞凋亡的关键蛋白酶caspase-3被激活,并且caspase-3酶活性与GLP浓度亦有剂量依赖性。提示GLP体外可诱导人肝癌HepG2细胞凋亡,其作用机制可能与bcl-2和survivin表达下调、p53表达上调及Caspase-3被激活有关。     

Emodin-induced apoptosis through p53-dependent pathway in human hepatoma cells

Most of the commonly used cytotoxic anticancer drugs have been shown to induce apoptosis in susceptible cells. However, the signaling pathway of their apoptotic effects remains undefined. In this study, the cytotoxic effect of emodin on various human hepatoma cell lines was investigated. Results demonstrated that emodin exhibited strongly suppressing effect on HepG2/C3A, PLC/PRF/5, and SK-HEP-1 cells, with the IC(50) value of 42.5, 46.6, and 53.1 microM, respectively. Furthermore, emodin induced apoptosis in HepG2/C3A cells was clearly verified by the appearance of DNA fragmentation and sub-G(1) accumulation. Besides, HepG2/C3A cells were found to be arrested in G(2)/M phase after the cells were treated with 60 microM emodin for 48 h. Moreover, significant increase in the levels of apoptosis-related signals such as p53 (419.3 pg/ml), p21 (437.4 units/ml), Fas (6.6 units/ml), and caspase-3 (35.4 pmol/min) were observed in emodin treated HepG2/C3A cells. Taken together, emodin displays effective inhibitory effects on the growth of various human hepatoma cell lines and stimulates the expression of p53 and p21 that resulted in the cell cycle arrest of HepG2/C3A cells at G(2)/M phase. Results also suggest that emodin-induced apoptosis in HepG2/C3A cells were mediated through the activation of p53, p21, Fas/APO-1, and caspase-3. It implies that emodin could be a useful chemotherapeutical agent for treatment of hepatocellular carcinoma (HCC).     

cAMP对转化细胞中几种基因表达及CREB DNA结合活性的影响

 从癌基因、抑癌基因及转录因子 CREB(c AMP反应序列结合蛋白 )对 CRE DNA序列结合活性的相关性 ,对 db- c AMP处理的小鼠 C3H10 T1 /2转化细胞增殖抑制作用进行了研究 .实验结果表明 ,转化细胞中 PKA(蛋白激酶 A)活性显著低于正常细胞 ,而 PKC(蛋白激酶 C)活性则显著高于正常细胞 .斑点印迹和 Northern印迹分析显示转化细胞中 c- myc和 Ca M(钙调素 )基因表达明显高于正常细胞 ,而 p53基因和 Rb基因表达则明显低于正常细胞 ,这些差别与 C3H10 T1/ 2 转化细胞增殖失控有关 .转化细胞经 db- c AMP(1 mmol/L)处理后 ,细胞增殖受到明显抑制 ,db- c AMP处理0 .5h后 ,转化细胞中 PKA活性便明显增强 ,PKC活性则被显著抑制 ,处理 2 h后 ,c- myc和 Ca M基因表达下降 ,而 p53和 Rb基因表达则增强 ,这些变化与 c AMP抑制 C3H10 T1/ 2 转化细胞增殖有密切联系 .凝胶阻滞电泳分析显示 db- c AMP(1 mmol/L )处理短时间内 ,CREB对 CRE DNA序列无结合活性 ,1 2 h后开始出现较弱的结合活性 ,2 4 h后才明显加强 ,表明在 db- c AMP处理的早期 ,调控区中含有 CRE序列的基因不参与 db- c AMP对细胞增殖抑制的调节 ,即与 CREB磷酸化及其相应的 DNA结合活性无相关性 .     

大鼠bcl-x_L cDNA的克隆和高滴度重组bcl-x_L腺相关病毒的制备

 以RT PCR法从大鼠脑组织中克隆bcl XL 基因 ,将其定向插入带rep cap基因和neu基因的三功能腺相关病毒 (AAV)载体 ,转染HeLa细胞并以G4 18筛选 ,然后用腺病毒感染筛选后的细胞克隆 ,包装成重组腺相关病毒 .用带rep cap基因的C12细胞筛选和滴定产生重组腺相关病毒的细胞克隆 ,粗制细胞裂解物中病毒滴度最高只有 3× 10 5IU ml.从产病毒量较高的克隆大量制备重组病毒 ,经肝素柱高压液相亲和层析法纯化、浓缩病毒后获得了高达 4× 10 11IU ml的重组病毒 .为研究脑缺血动物模型中BCL XL 的抗脑细胞凋亡作用打下了基础     

人乳腺癌MCF-7细胞系凋亡过程中bcl-2基因的结合蛋白

 Ｍ Ｃ Ｆ ７ 是 ｐ５３ 为野生型的人乳腺癌细胞系．为探讨该细胞系凋亡过程中 ｂｃｌ ２ 基因的下调机制,以 ５ 氟尿嘧啶（５ Ｆｕ）诱导凋亡,用 Ｐ Ｃ Ｒ 技术扩增出人 ｂｃｌ ２（ｈｂｃｌ ２）基因调控区长度为 ５５８ｂｐ 的片段,经亚克隆后的序列分析证实其准确性．用此 Ｐ Ｃ Ｒ 产物作为探针与 ５ Ｆｕ 刺激 １２ ｈ 后的 Ｍ Ｃ Ｆ ７ 核内蛋白质粗提物进行 Ｓｏｕｔｈｗ ｅｓｔｅｒｎ 印迹及凝胶阻滞（ Ｅ Ｍ Ｓ Ａ）分析．以未用 ５ Ｆｕ 刺激者为对照．结果显示,细胞核内一分子量为 ５３ ｋ Ｄ 的结合蛋白与 ｂｃｌ ２ 调控区的结合信号明显增强,而一种２０ ｋ Ｄ 的 Ｄ Ｎ Ａ 结合蛋白与该区的结合信号明显减弱．这些结果提示ｐ５３ 蛋白有可能是ｂｃｌ ２ 的负转录因子,２０ ｋ Ｄ 的 Ｄ Ｎ Ａ 结合蛋白有可能是ｂｃｌ ２ 的正转录因子．     

褪黑素通过提高p53和Fas的表达促进小鼠肝癌H22细胞凋亡

目的：研究褪黑素（MLT）对小鼠肝癌细胞株H22的促凋亡作用及其机理。方法：采用丫啶橙（AO）染色、培养液乳酸脱氢酶（LDH）活性检测和流式细胞术（FCM）观察MLT的促凋亡作用;采用RT-PCR方法检测MLT处理前后细胞的p53 mRNA、Fas mRNA的水平。结果：AO染色后H22细胞呈现明显核浓缩的凋亡形态;培养液LDH活性检测及FCM分析均提示MLT诱导H22细胞发生凋亡;RT-PCR结果显示p53、Fas表达增强。结论：MLT能促进H22细胞p53和Fas的表达,从而诱导细胞发生凋亡。     

Conformational and molecular basis for induction of apoptosis by a p53 C-terminal peptide in human cancer cells

A p53-derived C-terminal peptide induced rapid apoptosis in breast cancer cell lines carrying endogenous p53 mutations or overexpressed wild-type (wt) p53 but was not toxic to nonmalignant human cell lines containing wt p53. Apoptosis occurred through a Fas/APO-1 signaling pathway involving increased extracellular levels of Fas/FasL in the absence of protein synthesis, as well as activation of a Fas/APO-1-specific protease, FLICE. The peptide activity was p53-dependent, and it had no effect in three tumor cell lines with null p53. Furthermore, the C-terminal peptide bound to p53 protein in cell extracts. Thus, p53-dependent, Fas/APO-1 mediated apoptosis can be induced in breast cancer cells with mutant p53 similar to the recently described Fas/APO-1 induced apoptosis by wt p53. However, mutant p53 without p53 peptide does not induce a Fas/APO-1 activation or apoptosis. Docking of the computed low energy conformations for the C-terminal peptide with those for a recently defined proline-rich regulatory region from the N-terminal domain of p53 suggests a unique low energy complex between the two peptide domains. The selective and rapid induction of apoptosis in cancer cells carrying p53 abnormalities may lead to a novel therapeutic modality.     

Differential induction of apoptosis in x-irradiated L5178Y sublines bearing p53 mutation

We examined apoptosis and expression of p53, E2F-1, bax, bclx_L and bcl2 proteins in two L5178Y (LY) murine lymphoma sublines, LY-R and LY-S, which differ in radiosensitivity and double-strand break (DSB) repair. Both sublines are heterozygous for a p53 mutation in codon 170 that precludes the transactivation function. Accordingly, there is no G1/S arrest after irradiation.We found that there is no change in expression of E2F-1, bax, bclx_L or bcl2 proteins in both LY sublines after x-irradiation. LY-R cells do not constitutively express bcl2, whereas both sublines show high bax content. Radiation induces delayed apoptosis to a greater extent in LY-S than in LY-R cells. The apoptosis can be seen 24 h after irradiation (2 Gy) of LY-S cells, with a maximum at 48 h. LY-R cells need 5 Gy and 72 h post-irradiation incubation to show marked apoptosis (identified by the TUNEL method). The reported observations support the assumption that differential radiosensitivity of LY sublines is associated with the induction of apoptosis that is not related to transactivation by p53 and is primarily related to differential DNA repair ability. Received: 19 August 1999 / Accepted in revised form: 30 November 1999     

Wild-type function of the p53 tumor suppressor protein is not required for apoptosis of mouse hepatoma cells

The role of the tumor suppressor protein p53 in apoptosis of mouse hepatoma cells was studied. Different lines were used which were either p53 wild-type or carried various types of heterozygous or homozygous p53 mutations. The presence of mutations was demonstrated to correlate with a lack in transactivating activity of p53. While UV-light effectively produced apoptosis in cells of all lines, irrespective of their p53 mutational status, gamma-irradiation induced the formation of micronuclei but failed to induce apoptosis. Both UV- and gamma-irradiation led to nuclear accumulation and increases in p53 protein in p53 wild-type cells. Similarly, no significant differences in apoptotic response between p53 wild-type and p53 mutated cells were seen with other apoptotic stimuli like CD95/APO-1/Fas or TNFalpha. These data suggest that wild-type p53 is not required for induction of apoptosis in mouse hepatoma cells which may explain the apparent lack of p53 mutations in mouse liver tumors.     

Oxysterol-binding Protein-related Protein 8 (ORP8) Increases Sensitivity of Hepatocellular Carcinoma Cells to Fas-Mediated Apoptosis

Human hepatoma (HCC) has been reported to be strongly resistant to Fas-mediated apoptosis. However, the underlying mechanisms are poorly understood. In this study the function of oxysterol-binding protein-related protein 8 (ORP8) in human hepatoma cells apoptosis was assessed. We found that ORP8 is down-regulated, whereas miR-143, which controls ORP8 expression, is up-regulated in clinical HCC tissues as compared with liver tissue from healthy subjects. ORP8 overexpression triggered apoptosis in primary HCC cells and cell lines, which coincided with a relocation of cytoplasmic Fas to the cell plasma membrane and FasL up-regulation. Co-culture of HepG2 cells or primary HCC cells with Jurkat T-cells or T-cells, respectively, provided further evidence that ORP8 increases HCC cell sensitivity to Fas-mediated apoptosis. ORP8-induced Fas translocation is p53-dependent, and FasL was induced upon ORP8 overexpression via the endoplasmic reticulum stress response. Moreover, ORP8 overexpression and miR-143 inhibition markedly inhibited tumor growth in a HepG2 cell xenograft model. These results indicate that ORP8 induces HCC cell apoptosis through the Fas/FasL pathway. The role of ORP8 in Fas translocation to the plasma membrane and its down-regulation by miR-143 offer a putative mechanistic explanation for HCC resistance to apoptosis. ORP8 may be a potential target for HCC therapy.