产单核细胞李斯特菌诱导胸腺细胞凋亡及其基因调控

为研究产单核细胞李斯特菌 (LM )感染对小鼠胸腺细胞凋亡的诱导作用及胸腺细胞凋亡过程中的基因调控 ,小鼠经尾静脉注射LM后 ,以DNA琼脂糖凝胶电泳和流式细胞仪 (FCM )检测细胞凋亡及凋亡细胞的基因产物表达水平。结果表明LM能诱导小鼠胸腺细胞凋亡 ,琼脂糖凝胶电泳分析显示胸腺细胞出现典型的DNA“梯状带” ,FCM分析显示特征性的凋亡峰。胸腺细胞凋亡于LM (5× 10 5CFU )感染后 8h出现 ,48h达高峰。胸腺细胞凋亡百分率随LM感染剂量增加而增高。LM诱导小鼠胸腺细胞凋亡中p5 3、Bax及c myc基因表达产物明显增加 ,而Bcl 2基因表达产物水平无明显改变。提示LM以时间和剂量依赖方式诱导小鼠胸腺细胞凋亡 ,p5 3、Bax及c myc基因在LM诱导小鼠胸腺细胞凋亡的基因调控中起重要作用。     

白色念珠菌诱导小鼠胸腺细胞凋亡通路的探究

目的：探讨白色念珠菌诱导小鼠胸腺细胞凋亡的通路。方法：经小鼠尾静脉注射白色念珠菌后，用ELISA检测小鼠血清TNF-α水平；采用荧光分光光度计检测小鼠胸腺凋亡细胞caspase-3在不同时问组的活性变化；流式细胞仪检测小鼠胸腺细胞hax、p53、bcl-2基因产物水平。结果：TNF-α水平显著升高，caspase-3的活性明显增强，bax、p53基因表达上调。结论：白色念珠菌可能通过刺激机体TNF-α水平升高进而激活caspase-3，同时白色念珠菌上调bax、p53基因表达协同激活caspase-3，从而导致小鼠胸腺细胞凋亡。     

肝癌中细胞凋亡相关基因的表达及其意义

目的　为了探索肝癌发生中细胞凋亡相关基因所起的作用 ;方法 :利用免疫组化法 ,检测肝癌中bcl 2、bax和c myc基因的表达 ;结果 :肿瘤发生时 ,肝癌组织中bcl 2基因表达增强 ,抑制细胞凋亡 ;bax表达降低 ,促细胞凋亡的作用下降 ;c myc表达增强 ,其刺激细胞凋亡的作用可能消失。结论 :肝癌发生过程中 ,bcl 2、bax和c myc基因相互协调作用 ,细胞凋亡作用降低 ,使细胞增殖维持一定状态。     

硼替佐米上调fas、bcl2l12、caspase-9和caspase-3基因表达

目的： 探讨硼替佐米诱导慢性粒细胞白血病细胞株K562细胞凋亡及新基因bcl2l12在其中的作用。方法: MTT比色法观察硼替佐米对K562细胞的生长抑制作用;Annexin-V标记和线粒体跨膜电位（Δψm）分析细胞凋亡;RT-PCR方法检测0、6、12和24 h fas、bcl2l12、bcl-2、 bim、bax、caspase-3和caspase-9基因表达变化。结果: 硼替佐米抑制K562细胞生长呈时间和剂量依赖性,24 h和48 h半数抑制浓度分别为161.41 nmol/L和96.33 nmol/L;硼替佐米诱导K562细胞凋亡,12 h Annexin-V阳性细胞就开始增高,并呈时间依赖性,Δψm减低;RT-PCR显示fas、bcl2l12、caspase-3和caspase-9表达增高,但bcl-2、bim和bax表达无明显改变。结论: 硼替佐米可以抑制K562生长并诱导凋亡,上调fas、bcl2l12,使线粒体膜电位下降,激活caspase-9和caspase-3基因,促使DNA发生断裂可能是其诱导凋亡的机制之一。     

人IL-4基因修饰诱导肝母细胞瘤细胞凋亡及分化的研究

目的　研究人白细胞介素 4(IL 4)基因修饰对肝母细胞瘤细胞凋亡及分化的影响及可能机制。方法　以逆转录病毒为载体将人IL 4基因导入人肝母细胞瘤细胞系 (HepG2 )细胞。台盼蓝拒染、瑞氏染色、放射免疫测定、流式细胞仪细胞周期分析、原位杂交等方法检测人IL 4基因修饰后细胞形态、甲胎蛋白合成以及原癌基因c fos、c jun、c myc表达的变化。流式细胞仪AnnexinⅤ /PI双染色法及间接免疫荧光染色法检测凋亡细胞及凋亡调控基因p5 3、bcl 2的蛋白表达。结果　 (1)人IL 4基因修饰较空载体修饰及野生型HepG2细胞的细胞周期发生G0 /G1期阻滞 ,甲胎蛋白分泌量及原癌基因c fos、c jun、c myc表达降低 (P <0 .0 0 1)。细胞在形态及功能上趋向正常肝细胞转化 ;(2 )较空载体修饰及野生型HepG2细胞 ,IL 4基因修饰后部分细胞形态上出现核固缩等典型凋亡细胞的特征 ,流式细胞仪亦检测到 18.5 %± 4.7%的凋亡细胞 ;(3)人IL 4基因修饰增加p5 3而抑制bcl 2表达 (P <0 .0 5 )。结论　人IL 4基因修饰可诱导肝母细胞的凋亡及分化 ,诱导凋亡细胞可能与上调p5 3蛋白及抑制bcl 2蛋白表达有关。     

NO诱导血管平滑肌细胞凋亡原癌基因调控的初步研究

血管平滑肌细胞 (VSMC)异常增殖引起的细胞数量过度增加是动脉粥样硬化发生发展的一个主要病理特征 ,而 VSMC凋亡又在其中起着重要作用。引起细胞凋亡的因素有多种 ,近期发现 ,广泛存在于心血管系统的一氧化氮 (NO)除已知的多种生物作用外 ,还具有诱导巨噬细胞、VSMC凋亡的功能。但是 ,NO诱导 VSMC凋亡的调控机制尚不十分明确。而 bcl- 2、p5 3、bax、c- m yc、fas等都是参与细胞凋亡调控的原癌基因。鉴此 ,本实验探讨 NO诱导 VSMC凋亡与 bcl- 2、p5 3、bax之间的关系。方法 :采用贴块法培养大鼠主动脉 VSMC,3～ 8代细胞用于实…     

硼替佐米上调fas、bcl2112、caspase-9和caspase-3基因表达

目的：探讨硼替佐米诱导慢性粒细胞白血病细胞株K562细胞凋亡及新基因bcl2H2在其中的作用。方法：MTT比色法观察硼替佐米对K562细胞的生长抑制作用;Annexin—V标记和线粒体跨膜电位（△ψm）分析细胞凋亡;RT—PCR方法检测0、6、12和24h fas、bcl2112、bcl-2、bim、bax、caspase-3和caspase-9基因表达变化。结果：硼替佐米抑制K562细胞生长呈时间和剂量依赖性,24h和48h半数抑制浓度分别为161．41nmol／L和96．33nmol／L;硼替佐米诱导K562细胞凋亡,12hAnnexin—V阳性细胞就开始增高,并呈时间依赖性,AtOm减低;RT—PCR显示fas、bcl2112、caspase-3和caspase-9表达增高,但bcl-2、bim和bax表达无明显改变。结论：硼替佐米可以抑制K562生长并诱导凋亡,上调如、bcl2112,使线粒体膜电位下降,激活caspase-9和caspase-3基因,促使DNA发生断裂可能是其诱导凋亡的机制之一。     

细胞凋亡的分子机制及其调控因子

自 1972年Kerr提出凋亡概念以来 ,有关凋亡的生理和病理研究逐渐深入广泛 ,研究发现细胞凋亡是多细胞生物生命活动中不可缺少的组成内容 ,凋亡既与机体组织的发育、正常生理活动的维持关系密切 ,又与肿瘤、病毒感染和自身免疫性疾病的发生密切相关。现已发现Fas、bcl 2、p5 3、c myc、ced、TGF β等基因皆是参与调控细胞凋亡的重要基因。本文就细胞凋亡及其调控因子研究发现和进展综述如下     

bcl—2／bax p53c—myc在肾盂和输尿管肿瘤中的表达及意义

越来越多研究表明肿瘤的发生发展与多个基因有关，细胞凋亡相关基因bcl—2／bax、p53和c—myc基因与多种肿瘤的产生和发展有关。许多研究表明在肾盂、输尿管肿瘤中检测这些基因的表达对肿瘤的诊断、分期和预后有重要意义，本文就此方面的研究现状作一综述。     

全国第六届组织学与胚胎学青年学术研讨会论文摘要(续)

73.NO诱导血管平滑肌细胞凋亡原癌基因调控的初步研 究田雪梅李进第一军医大学组织学胚胎学教研室 广州 510515 血管平滑肌细胞(VSMC)异常增殖引起的 细胞数量过度增加是动脉粥样硬化发生发展的一个主要病 理特征,而VSMC凋亡又在其中起着重要作用.引起细胞凋 亡的因素有多种,近期发现,广泛存在于心血管系统的一氧 化氮(NO)除已知的多种生物作用外,还具有诱导巨噬细胞、 VSMC凋亡的功能.但是,NO诱导VSMC凋亡的调控机制 尚不十分明确.而bcl-2、p53、bax、c-myc、fas等都是参与细 胞凋亡调控的原癌基因.鉴此,本实验探讨NO诱导VSMC 凋亡与bcl-2、p53、bax之间的关系.方法:采用贴块法培养大 鼠主动脉VSMC,3～8代细胞用于实验.以亚硝基乙酰青霉 胺(SNAP)作为NO供体.构建逆转录病毒载体,将bcl-2基 因转入VSMC,使VSMC中bcl-2蛋白过量表达,通过原位 末端标记和流式细胞术检测VSMC在SNAP作用下的凋 亡.并采用免疫荧光染色,借助粘附式细胞仪(ACAS507)检 测细胞中bcl-2、p53、bax原癌蛋白表达的变化.结果:通过转 染bcl-2基因能使VSMC中bcl-2蛋白过量表达,且能使 SNAP作用下VSMC的凋亡率减少,而细胞中p53、bax蛋 白表达在转染和未转染bcl-2的VSMC中无明显差异.结 论:bcl-2过量表达阻止NO诱导的VSMC凋亡,但此作用 不是通过减少细胞中p53、bax蛋白的表达,而可能是通过增 加Bcl-2/bax比例的方式实现的.     

c-fos反义寡聚核苷酸对亚硒酸钠引起的皮质神经元凋亡的保护作用(英文)

本文利用 c-fos ASO(反义链全部硫代磷酸化修饰 )技术阻断 c-fos的表达 ,观察此项处理对亚硒酸钠在体外培养皮质神经细胞致凋亡作用和相关基因表达改变的影响。结果显示 :( 1) c-fos ASO可阻断亚硒酸钠的致凋亡作用 ,且有一定的剂量和时间依赖性 ;用琼脂糖凝胶电泳分析由各个时间点提取的 DNA片段 ,与对照组比较 ,c-fos ASO能明显阻断亚硒酸钠诱导的典型的 DNA梯型 ;用流式细胞仪检测得到的 DNA含量直方图也显示 ,在 c-fos ASO处理后 ,与对照组比较 ,亚硒酸钠引起的亚二倍体峰变小 ;( 2 )用 RT-PCR法检测表明 ,在用 c-fos ASO和亚硒酸钠处理皮质神经细胞后 ,不仅阻断了 c-fos表达的上调 ,对其它几种基因表达的改变也有不同的影响 ,与对照组比较 ,c-fosm RNA在各个时间点 ( 0 .5、1、2、4和 2 4h)不再上升 ,bcl-2 m RNA则不再下降 ,bax m RNA和 ACh E m RNA都不再上升 ;唯一不同的是 ,p5 3m RNA的变化趋势则与未经 c-fos ASO预处理时基本相同 ,在多数时间点两组 p-5 3m RNA水平之间无显著性差异 ( P>0 .0 5 ) ,说明此基因的表达基本不受 c-fos ASO作用的影响。上述结果提示 ,亚硒酸钠诱导的神经元凋亡可能是一组级联式基因表达更变的结果 ,c-fos是其中的一个组成成员 ,它的表达改变被阻断后 ,处于它下游?     

不同胎龄的胎儿和少儿皮肤中bax,bcl-2和p53基因表达的变化

目的：探讨凋亡相关基因bax, bcl-2和p53在不同胎龄的胎儿皮肤和少儿皮肤组织中表达的变化特征及其可能的生物学意义。方法： 运用末端脱氧核糖转移酶介导的生物素化脱氧尿嘧啶缺口标记技术(TUNEL)检测18例不同胎龄(13-32周)的胎儿皮肤和6例少儿皮肤组织中细胞凋亡的变化后，提取这些皮肤组织中的总RNA，分离mRNA，用RT-PCR方法检测bax, bcl-2和p53基因在不同组织中的表达变化特征。结果： 随着胎儿的生长发育，皮肤组织中的细胞凋亡率逐渐增加。在早期妊娠胎儿的皮肤中，bcl-2基因表达水平较高，随着胎龄的增加，bcl-2基因的转录本含量逐渐降低，在少儿的皮肤组织中，这种基因的表达量明显低于早期妊娠胎儿皮肤（P<0.01）。与bcl-2基因不同，在早期妊娠胎儿皮肤组织中，p53基因表达水平较低，而在晚期妊娠胎儿和少儿的皮肤内，该基因表达较强，而bax基因在不同发育时期的胎儿和少儿皮肤组织中表达差异不显著（P>0.05）。结论： 晚期妊娠胎儿和少儿皮肤组织中细胞增殖减缓，细胞趋向分化或凋亡的增加可能与p53基因表达增强，bcl-2表达降低相关；而p53表达降低，bcl-2表达升高可能是早期妊娠胎儿皮肤中细胞凋亡较少的机制之一。     

Expression of the apoptosis-related oncogenes bcl-2, bax, and p53 in Merkel cell carcinoma: can they predict treatment response and clinical outcome?

Chemotherapy and radiation therapy act predominantly through the induction of apoptosis in malignancies. Merkel cell carcinoma, an aggressive malignancy with prominent apoptosis, has proved to be sensitive to both modes to a certain degree. We used immunohistochemical methods to examine 25 Merkel cell carcinomas and 8 of their lymph node metastases to assess the status of the antiapoptotic gene bcl-2 and 2 proapoptotic genes, wild-type p53 and bax. All tumors showed prominent bax immunopositivity; 76% were positive for bcl-2, and only 28% were positive for p53, the latter presumably reflecting mutated p53. No statistically significant relationship was found between tumor immunopositivity and therapy response or survival. The widespread bax immunopositivity and the apparently low rate of p53 mutations, as suggested by the low rate of p53 immunopositivity, may be related to the presence of prominent apoptosis in Merkel cell carcinoma. The finding of bcl-2 immunopositivity in 76% of the tumors suggests that some of the tumor cells may be resistant to apoptosis-inducing agents.     

Bax is an important determinant for radiation sensitivity in esophageal carcinoma cells

Apoptosis is a crucial phenomenon for radiation-induced cell death. Since Bax plays a critical role in inducing apoptosis via p53-dependent and -independent pathways, we analyzed a role of Bax in radiation sensitivity in esophageal carcinoma cells. Using eight human esophageal carcinoma cell lines, irradiation was performed with cobalt-60 (60Co) gamma-rays. Radiation sensitivity was determined by induction of apoptosis, which was assessed by morphological change in nuclear condensation of chromatin, DNA ladder formation and apoptosis-related genes after irradiation. The survival curve was evaluated by clonogenic assay using a parameter D0 after irradiation, compared to that of the control. After transfection of the bax gene into low radiation sensitivity, TE-1 cells were conducted by lipofection method using pSFFV-Neo vector carrying bax cDNA. Radiation sensitivity of esophageal carcinoma cells was associated with induction of apoptosis, in a time- and dose-dependent manner. Induction of apoptosis affects early responsiveness to irradiation rather than the parameter D0. Radiation-induced apoptosis was associated with an increase in expression of bax gene, regardless of p53 genetic status. The introduction of the bax gene into a low radiation sensitivity cell line, TE-1, enhanced radiation sensitivity in association with increased apoptotic cell death after irradiation. Radiation sensitivity of esophageal carcinoma cells can be evaluated by induction of apoptosis, as an early predictive marker for radiation response. The proapoptotic gene bax plays a critical role in the determination of tumor response in radiation therapy.     

抗阻训练衰老小鼠骨骼肌p53与bax基因的表达

背景：抗阻训练能够缓解肌肉衰减征的发生并能够弱化骨骼肌细胞凋亡。 目的：观察年龄和抗阻训练对SAMP8小鼠骨骼肌p53,bax基因表达的影响。 方法：分别取3月龄(青年组)和6月龄(老年组)的SAMP8小鼠进行8周爬梯运动,每周3次,以不参加运动的同龄小鼠作为对照。 结果与结论：Real-time PCR结果显示,与青年组比较,老年组小鼠骨骼肌纤维p53,bax mRNA的表达量显著上升(P < 0.05),而与相同月龄对照组比较,抗阻训练的小鼠骨骼肌纤维中p53,bax mRNA的表达量明显降低(P < 0.05)。提示抗阻训练能抑制衰老小鼠骨骼肌p53,bax基因的表达。     

bcl—2／bax对不同乳腺癌细胞凋亡的调节作用

目的：了解bcl-2/bax在乳腺癌细胞的p53依赖性凋亡和p53非依赖性凋亡中的作用。方法：用原位杂交的方法检测化疗药物VM-26诱导入乳腺癌细胞株MCF-7和MDA-MB-435S凋亡前后bcl-2和bax的mRNA表达情况。结果：与MDA-MB-435S细胞相比,MCF-7细胞的bcl-2 mRNA水平较高。两株细胞均能在VM-26的作用下发生凋亡。药物诱导后,MCF-7细胞的bcl-2 mRNA表达下降,bax mRNA表达增加;MDA-MB-435S细胞bax mRNA表达增加,bcl-2、mRNA无明显变化。结论：wt p53可在转录水平调节bcl-2/bax的表达,从而介导凋亡作用;bax在p53非依赖性凋亡中也发挥一定作用;bcl-2的高表达可能与肿瘤耐药性有关。     

Possible involvement of bcl-2 suppression in wild-type p53 gene-dependent cell growth repression in rat osteosarcoma cells

We recently obtained 3 cloned cell lines demonstrating the p53 mutation from a lung metastatic nodule of a rat transplantable osteosarcoma. In this study, we applied wild-type p53 gene transfer to the rat osteosarcoma cells by lipofection to investigate the effects on cell growth, expression of genes such as waf1/p21, bcl-2, and bax, and nucleosomal DNA fragmentation due to apoptosis. Reconstitution of the p53 gene inhibits cellular growth, and this growth-suppressive effect is partly due to apoptosis involving bcl-2 gene suppression in this tumor type. This rat osteosarcoma model is similar in biologic behavior to human cases and thus is very suitable for further investigation of tumorigenesis and gene therapy for osteosarcoma.     

Nitric oxide induces murine thymocyte apoptosis by oxidative injury and a p53-dependent mechanism.

Previously, we showed that NO induces thymocyte apoptosis via a caspase-1-dependent mechanism [(1) ]. In the present study, we investigated the role of heme oxygenase, catalase, bax, and p53 in this process. The NO donor, S-nitroso-N-acetyl penicillamine (SNAP), induced DNA fragmentation in thymocytes in a time- and concentration-dependent way. SNAP (100 microM) induced 50--60% apoptosis; higher doses did not increase the rate of apoptosis significantly. SNAP decreased catalase and heme iron (Fe) levels without affecting superoxide dismutase, glutathione, or total Fe stores in thymocytes. SNAP significantly increased the expression of heme oxygenase 1 (HSP-32), p53, and bax but not bcl-2. Treatment with the heme oxygenase inhibitor, tin protoporphyrin IX inhibited SNAP-induced thymocyte apoptosis. Furthermore, thymocytes from p53 null mice were resistant to NO-induced apoptosis. Our data suggest that NO may induce its cytotoxic effects on thymocytes by modulating heme oxygenase and catalase activity as well as up-regulating pro-apoptotic proteins p53 and bax.     

Protein expression of p53, p21 (WAF1/CIP1), bcl-2, Bax, cyclin D1 and pRb in human colon carcinomas

Tumour growth is regulated by a balance between proliferation, growth arrest and programmed cell death (apoptosis). Until recently, the majority of the studies dealing with oncogenesis has been focused on the regulation of cell proliferation. There is now growing understanding that control of growth arrest and apoptosis play key roles in the development of human cancer and in cancer treatment. Some of the more heavily studied proteins of importance for the control of growth arrest and apoptosis are p53, p21, bcl-2 and bax. Alterations in the p53 protein may lead to malignant transformation and defect therapy response, most likely as a result of defective p53-dependent apoptosis. In addition, p21 (WAF1/CIP1) is involved in cell-cycle arrest and probably in induction of p53-dependent apoptosis. Proteins belonging to the bcl-2 family are also important for normal apoptosis. Overexpression of bcl-2 protein is thought to reduce the apoptotic capacity, while bax protein seems to be necessary for induction of apoptosis. In this study, we have immunostained tissues from 93 primary colon carcinomas and have examined the expression of p53, p21 (WAF1/CIP1), bcl-2 bax, pRb and cyclin D1 for evaluation of their roles in colon-cancer progression. A highly significant association between p53 accumulation and downregulation of p21 (WAF1/CIP1) was seen. We also found a strong association between reduced/absent p21 and the development of metastases and death due to cancer disease. Cyclin D1, bcl-2 and bax protein failed to have independent prognostic impacts. Bcl-2 and bax protein levels showed an inverse relationship. The results of the present study indicate that reduced p21 protein levels play an important role in progression of colon cancer. We concluded that evaluation of p21 expression in primary colon carcinomas at the time of surgery might be a valuable tool in defining patients with a high risk of developing metastases. Received: 22 June 1999 / Accepted: 24 September 1999