Expression ofbcl-2 gene increases in apoptosis of vascular endothelial cells induced by rattlesnake venom

Apoptosis of vascular endothelial cells (VEC) has been induced by deprivation of survival factors (aFGF and serum) and by rattlesnake venom. The expression ofbcl-2 gene has been examined by Northern blotting in the two apoptosis inducing systems. Our results show that the expression ofbcl-2 has not been detected in normal culture cells and in apoptotic cells induced by deprivation of survival factors. But in apoptotic cells induced by rattlesnake venom (10 ng/mL), the expression ofbcl-2 increases, and its mRNA exhibits two bands. The data first suggest that increasing expression and splitting ofbcl-2 mRNA may play an important role in apoptosis of VEC induced by rattlesnake venom, and this finding is helpful to understanding the role ofbcl-2 in regulation of apoptosis.     

血管内皮细胞凋亡过程中几种癌基因表达的研究

为了研究细胞凋亡的分子调控机制 ,用光学显微技术、DNA凝胶电泳和Northernblot方法 ,研究了去除生长因子 (FGF和血清 )和蛇毒诱导的两个血管内皮细胞凋亡系统中 p53、c H ras、c myc和bcl 2基因的表达 .发现去除生长因子诱导的细胞凋亡过程中 ,p53基因表达显著增加 ,c H ras和c myc基因表达无变化 ;蛇毒诱导细胞凋亡过程中 ,p53基因表达显著增加 ,c H ras和c myc基因表达无变化 .在正常生长和凋亡细胞中均未检测到bcl 2基因的明显表达 .实验结果表明 :p53基因参与上述两种细胞凋亡诱导系统的分子调控 ;c H ras基因只参与去除生长因子诱导的细胞凋亡过程 ,而不参与蛇毒诱导的细胞凋亡过程 ;这两种细胞凋亡诱导系统均与c myc基因表达无关 ;未见bcl 2基因明显参与血管内皮细胞的凋亡过程 .     

一个新的靶点的反义bcl—2寡核苷酸诱导HL—60细胞凋亡的研究

目的：探讨针对bcl-2mRNA蛋白编码区的反义寡核苷酸对HL-60细胞bcl-2蛋白表达和凋亡的作用。方法：应用台盼蓝拒染法和流式细胞仪检测HL-60细胞的活性和bcl-2蛋白表达和细胞凋亡的情况。结果：10μmol/L和20μmol/L的反义寡核苷酸能抑制HL-60细胞bcl-2蛋白表达和诱导细胞调亡,并且针对bcl-2mRNA蛋白编码区的反义寡核苷酸比针对bcl-2mRNA翻译起始区的反义寡核苷酸作用更强。结论：针对bcl-2mRNA蛋白编码区的反义寡核苷酸能抑制HL-60细胞bcl-2蛋白表达和诱导细胞调亡。     

Apoptotic cell death induced by c-myc is inhibited by bcl-2.

Apoptosis is a form of physiological cell death, characterized by chromatin condensation, cytoplasmic blebbing and DNA fragmentation, which often depends on RNA and protein synthesis by the dying cell. The c-myc proto-oncogene, usually implicated in cell transformation, differentiation and cell-cycle progression also has a central role in some forms of apoptosis. These opposing roles of myc in cell growth and death require that other gene products dictate the outcome of c-Myc expression on a cell. A candidate for such a modifying gene is bcl-2, whose product prolongs cell survival and blocks apoptosis in some systems. Here we demonstrate that Bcl-2 prevents apoptotic death induced by c-Myc, provide a mechanism whereby cells can express c-Myc without undergoing apoptosis, and give a possible explanation for the ability of Bcl-2 to synergize with c-Myc in cell transformation.     

小剂量阿糖胞苷体外诱导HL-60细胞凋亡的作用

阿糖胞苷是白血病的重要药物,其小剂量化疗仍用于临床治疗白血病,并取得了一定的疗效,但其作用机制尚未完全阐明,可能与药物诱导细胞分化或凋亡有关.文中用TUNEL法和流式细胞仪分析小剂量Ara-C(10-8M)对HL-60细胞周期和细胞凋亡的影响,用免疫组化法和原位杂交法分别检测P53蛋白和p53mRNA、bcl-2mRNA表达水平的改变,以探讨bcl-2和p53基因表达与小剂量阿糖胞苷诱导HL-60细胞凋亡的关系.结果表明:小剂量Ara-C能诱导HL-60细胞凋亡,其分子机制可能与P53蛋白、p53mRNA表达增加和bcl-2mRNA表达降低有关.     

Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death

The t(14; 18) chromosomal translocation of human follicular B-cell lymphoma juxtaposes the bcl-2 gene with the immunoglobulin heavy chain locus. The bcl-2 immunoglobulin fusion gene is markedly deregulated resulting in inappropriately elevated levels of bcl-2 RNA and protein. Transgenic mice bearing a bcl-2 immunoglobulin minigene demonstrate a polyclonal expansion of resting yet responsive IgM-IgD B cells which display prolonged cell survival but no increase in cell cycling. Moreover, deregulated bcl-2 extends the survival of certain haematopoietic cell lines following growth-factor deprivation. By using immunolocalization studies we now demonstrate that Bcl-2 is an integral inner mitochondrial membrane protein of relative molecular mass 25,000 (25k). Overexpression of Bcl-2 blocks the apoptotic death of a pro-B-lymphocyte cell line. Thus, Bcl-2 is unique among proto-oncogenes, being localized to mitochondria and interfering with programmed cell death independent of promoting cell division.     

强力霉素对人非小细胞肺癌A549细胞的增殖抑制及促凋亡作用

为研究强力霉素体外对人非小细胞肺癌A549细胞的增殖及凋亡的影响。应用MTT比色法研究不同浓度强力霉素(2.5,5,10,20,50mg/L)对A549细胞增殖的影响。荧光显微镜、琼脂糖凝胶电泳、AnnexinV-PI双标法等多项方法,研究强力霉素对A549细胞的促凋亡作用;采用免疫组化的方法,观察药物作用后bcl-2和bax的表达。强力霉素作用后,A549细胞的吸光度值降低,并出现凋亡特有的细胞核改变DNA梯状条带,早期凋亡的细胞数增加,伴有bcl-2的下调,bax上调。这表明强力霉素可抑制人非小细胞肺癌A549细胞的生长,并诱导细胞发生凋亡,其诱导凋亡的作用可能与其调节bcl-2/bax的表达有关。     

白介素24对单个核细胞bcl-2表达的影响

为探讨白细胞介素24(IL-24)对成人急性白血病骨髓单个核细胞(BMMNC)体外生长的影响,并初步研究其作用机制。随机选取急性白血病住院患者15例,均经FAB分型或免疫学分型确诊,其中确诊未治的成人急性淋巴细胞白血病(ALL)患者5例,成人急性非淋巴细胞白血病(ANLL)患者10例。取患者骨髓,分离单个核细胞并培养,将不同浓度IL-24作用于BMMNC。应用四氮唑蓝(MTT)比色还原法、AnnexinV-FITC染色流式细胞术及逆转录-聚合酶联反应(RTPCR),分别检测IL-24对急性白血病BMMNC体外细胞增殖与凋亡及bcl-2表达的影响。结果显示,IL-24对体外培养的白血病BMMNC有明显的生长抑制作用,且呈时间、剂量依赖性,与阴性对照组相比差异有统计学意义(P0.05)。IL-24浓度50 ng/mL作用急性白血病BMMNC,48 h后凋亡率为39.21%±5.79%,高于对照组(P0.01)。不同浓度IL-24处理白血病BMMNC,能够降低bcl-2 mRNA的表达。IL-24体外培养的成人急性白血病BMMNC,有明显的生长抑制、诱导凋亡作用。由此可知,IL-24可以下调bcl-2 mRNA表达,可能是IL-24抑制急性白血病BMMNC生长作用的重要机制。     

Construction and application of L929 cell model expressing human bcl-2 protein

Recombinant eucaryotic expression vector pLXSN/s-bcl-2 has been constructed by cloning human bcl-2 cDNA containing the full-length open reading frame into the vector pLXSN in sense orientation, and a mammalian cell model expressing human bcl-2 protein has been established by electroporating the recombinant vector into mouse L929 cells. bcl-2 expression in L929 cells has no effect on the cell growth and survival under normal culture conditions, but it can enhance the survival of the cell in the challenge of some apoptosis-inducing stimuli, including tumor necrosis factor α(TNF α) and staurosporine (STS).     

运动对心肌细胞中凋亡调控基因的影响

研究运动对心肌细胞中调控基因Bcl-2、Bax和p53的影响以探讨凋亡调控基因对运动引起心肌细胞凋亡的作用。以大鼠中等运动强度训练、一次性力竭运动和过度训练为运动模型，采用反转录聚合酶链式反应（RT-PCR）技术观察了大鼠心肌细胞中调控基因Bcl-2、Bax和p53mRNA的表达。长期中等强度的运动可造成大鼠心肌细胞中凋亡调控基冈Bcl-2mRNA表达明显增加，可抑制心肌细胞凋亡；而力竭运动和过度训练可引起心肌细胞中Bcl-2mRNA表达下降和调控基冈Bax、p53mRNA表达显著升高以及凋亡调控基因Bcl-2／Bax比值显著下降，可促进心肌细胞凋亡。心肌细胞中凋亡调控基冈Bcl-2、Bax和p53在不同运动后的不同表达对心肌细胞凋亡的发生有明显的调控作用。     

JAK-STAT/survivin通路对过氧化氢预处理诱导的适应性细胞保护作用的影响

为了探讨H_2O_2预处理对存活素(survivin)表达的影响及JAK-STAT/survivin通路在H_2O_2预处理的适应性细胞保护中的作用,用PC12细胞建立H_2O_2预处理对抗H_2O_2诱导细胞凋亡的实验模型,应用甲氮甲哇蓝(MTT)法检测细胞存活率,碘化丙啶(PI)染色流式细胞术检测细胞凋亡率,Hoechst染色检测细胞凋亡的形态学改变,免疫印迹法(Western blot)测定survivin表达水平.结果显示:用100μmol/L H_2O_2预处理PC12细胞90min可明显地抑制300μmol/L H_2O_2作用12 h后引起的细胞毒性和细胞凋亡,并可以显著地促进PC12细胞survivin的表达;JAK_2抑制剂AG-490不仅可以抑制survivin的表达.而且能拮抗H_2O_2预处理的适应性细胞保护作用.研究表明,survivin是JAK-STAT通路的靶基因,JAK-STAT/survivin通路在H_2O_2预处理诱导的适应性细胞保护机制中起着重要的作用.     

Effect of thrombin on the apoptosis of hippocampal neurons in vitro

Hippocampal neurons were treated by thrombin and thrombin receptor activating peptides (TRAP). Cell survival rate was decreased in a dose-dependent manner by MTT assay. The numbers of apoptotic cell and apoptotic rate of hippocampal neurons treated by different concentrations of thrombin were increased in a dose-dependent manner by terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end-labeling (TUNEL) method and Flow Cytometry. When the concentration of thrombin is 40 U/mL, TUNEL positive cells and apoptotic rate of hippocampal neurons reached peak value, were 27.3±4.0 and (29.333±4.633)%, respectively. Immunocytochemistry assay show that Bcl-2 protein expression was down-regulated and Bax protein expression was up-regulated with the concentration of thrombin increased. TRAP can mimic the effect of thrombin to induce apoptosis on hippocampal neurons. These data demonstrated that thrombin induced hippocampal neuron apoptosis in a dose-dependent manner through activating protease-activated protein-1 (PAR-1). The change in expression of Bcl-2 and Bax was related with the effect of high concentration thrombin induced apoptosis on hippocampal neurons. Foundation item: Supported by the Natural Science Foundation of Hainan Province (N30215) Biography: YANG Wen-qiong (1968-), female, Ph.D. candidate, research direction: cerebrovascular disease.     

PF18-3, a 35-ku molecule, expresses specifically on apoptotic subset of mouse thymocytes

PF18-3 monoclonal antibody (mAb), one of the rat mAbs against mouse thymic stromal cells (MTSC), has been found to inhibit thymocyte apoptosis induced by a mouse thymic dendritic cell line, MTSC4, in previous co-culture study. The aim of this research is to investigate the character of PF18-3 rnAb recognized molecule (PF18-3 molecule) and its role in MTSC4-induced thymocyte apoptosis. The characterization of PF18-3 molecule expression has been conducted by FACS analysis. PF18-3 molecules have been found to express on MTSC4 as well as on Con A activated but not freshly isolated thyrnocytes. Up-regulated expression of PF18-3 molecules has been also observed on thymocytes after being co-cultured with MTSC4 for 48 h. The results from FACS analyses by PI staining for detecting apoptosis-related hypodiploid and by PF18-3 rnAb staining reveal that PF18-3 molecules expresss specifically on the apoptotic subgroup of thymocytes with high hypodiploid content. The PF18-3 molecule expressed on apoptotic thymocytes with 35 ku of molecular weight, identified by immunoprecipitation and western blotting, is thus likely to be a molecule involved in thymocyte apoptosis.     

PF18-3, a 35-ku molecule, expresses specifically on apoptotic subset of mouse thymocytes

PF18-3 monoclonal antibody (mAb), one of the rat mAbs against mouse thymic stromal cells (MTSC), has been found to inhibit thymocyte apoptosis induced by a mouse thymic dendritic cell line, MTSC4, in previous co-culture study. The aim of this research is to investigate the character of PF18-3 mAb recognized molecule ( PF18-3 molecule) and its role in MTSC4-induced thymocyte apoptosis. The characterization of PF18-3 molecule expression has been conducted by FACS analysis. PF18-3 molecules have been found to express on MTSC4 as well as on Con A activated but not freshly isolated thymocytes. Up-regulated expression of PF18-3 molecules has been also observed on thymocytes after being co-cultured with MTSC4 for 48 h. The results from FACS analyses by PI staining for detecting apoptosis-related hypodiploid and by PF18-3 mAb staining reveal that PF18-3 molecules expresss specifically on the apoptotic subgroup of thymocytes with high hypodiploid content. The PF18-3 molecule expressed on apoptotic thymocytes with 35 ku of molecular weight, identified by immunoprecipitation and western blotting, is thus likely to be a molecule involved in thymocyte apoptosis.     

TPA诱导肝癌细胞SMMC7721凋亡及与RXRα和TR3的关系

 在肝癌细胞SMMC7721中研究TPA诱导的凋亡及它与细胞核受体TR3和RXRα的关系.实验中使用SMMC7721肝癌细胞.用MTT法检测生长抑制.用DAPI荧光染色法观察凋亡形态,随机记数1 000个细胞以计算凋亡指数.用CAT分析检测RXRα转录活性的抑制.用Western Blot检测RXRα蛋白的表达水平.用RT-PCR检测TR3 mRNA的表达水平.用MTT检测到TPA对SMMC7721肝癌细胞的生长抑制,并且有剂量依赖性.TPA诱导SMMC7721肝癌细胞的凋亡,RXRα蛋白随时间依赖性的降解,并且用CAT分析检测到TPA能抑制RXRα的转录活性.另外TPA能诱导TR3 mRNA的表达上调.TPA抑制SMMC7721肝癌细胞的生长,并诱导凋亡.TPA能上调TR3的mRNA,使RXRα随时间依赖性的降解,并能抑制RXRα的转录活性.因此TPA诱导SMMC7721肝癌细胞的凋亡可能与细胞核受体TR3和RXRα有某些联系.     

博莱霉素肺损伤大鼠细胞凋亡及bcl—2／bax基因的表达

目的：探讨细胞凋亡与博莱霉素诱导大鼠早期肺纤维化及bax,bcl-2对博莱霉素大鼠肺纤维化细胞凋亡的作用。方法：通过复制博莱霉素大鼠肺纤维化模型,采用TUNEL、免疫组织化学、原位杂交及透射电镜等技术,观察博莱霉素大鼠肺组织细胞凋亡的变化及bax,bcl-2,bcl-2mRNA和蛋白的表达。结果：博莱霉素诱导的大鼠肺纤维化细胞凋亡明显增加,肺上皮细胞bax蛋白表达明显上调,bcl-2mRNA及蛋白表达明显减弱,间质细胞bcl-2mRNA及蛋白表达增加。结论：肺组织细胞凋亡及bcl-2表达下调可能参与博莱霉素诱导大鼠肺纤维化的发生,凋亡相关基因bax,bcl-2mRNA及蛋白参与博莱霉素诱导大鼠肺纤维化细胞凋亡的调控。     

siRNA对肺癌细胞株NCI-H460 bcl-2基因表达的影响

目的:研究siRNA (smallinterferenceRNA)对大细胞肺癌细胞株NCI -H4 6 0bcl- 2基因表达的影响。方法:利用Ambion公司提供的设计软件和试剂盒,设计合成以bcl - 2基因为靶标的siRNA ,通过脂质体将合成的siRNA转入NCI-H4 6 0细胞株,设置转染bcl- 2反义药物G3139和空白两对照组。用MTT法检测siRNA对细胞生长的作用;流式细胞仪检测细胞周期的改变和Bcl- 2蛋白表达;RT -PCR检测bcl- 2mRNA水平。结果:siRNA组与对照组细胞存活率均有显著性差异(P <0 0 5 ) ;siRNA组bcl- 2的mRNA明显低于对照组和反义组(P <0 0 5 ) ;siRNA组Bcl- 2蛋白阳性率明显低于对照组和反义组,siRNA组以及反义组细胞阻滞于S期。结论:体外转录合成的siRNA可抑制NCI-H4 6 0细胞bcl- 2基因的表达,抑制率可达5 0 %以上。     

敬钊缨毛蛛蛛毒对人肝癌细胞BEL-7402细胞增殖、细胞周期和C-myc蛋白的影响

通过MTT方法可知敬钊缨毛蛛蛛毒对BEL 7402细胞增殖有较强的抑制作用(p<0.05),时效和量效关系良好.IC50为18mg/L.敬钊缨毛蛛蛛毒可以抑制BEL 7402细胞DNA的合成.流式细胞仪检测发现经过蛛毒作用的BEL 7402细胞凋亡率增加,细胞周期阻滞在G0/G1期.WesternBlot方法进一步检测到蛛毒作用BEL 7402细胞72h后,C myc蛋白表达减弱.实验结果表明,敬钊缨毛蛛蛛毒可以抑制人肝癌细胞BEL 7402的增殖和DNA的合成,其药理作用机理可能除了诱导凋亡外主要是使BEL 7402细胞周期相关蛋白C myc表达减弱,导致细胞周期的变化.     

Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells

A common feature of follicular lymphoma, the most prevalent haematological malignancy in humans, is a chromosome translocation (t(14;18] that has coupled the immunoglobulin heavy chain locus to a chromosome 18 gene denoted bcl-2. By analogy with the translocated c-myc oncogene in other B-lymphoid tumours bcl-2 is a candidate oncogene, but no biological effects of bcl-2 have yet been reported. To test whether bcl-2 influences the growth of haematopoietic cells, either alone or together with a deregulated c-myc gene, we have introduced a human bcl-2 complementary DNA using a retroviral vector into bone marrow cells from either normal or E mu-myc transgenic mice, in which B-lineage cells constitutively express the c-myc gene. Bcl-2 cooperated with c-myc to promote proliferation of B-cell precursors, some of which became tumorigenic. To determine how bcl-2 expression impinges on growth factor requirements, the gene was introduced into a lymphoid and a myeloid cell line that require interleukin 3 (IL-3). In the absence of IL-3, bcl-2 promoted the survival of the infected cells but they persisted in a G0 state, rather than proliferating. These results argue that bcl-2 provided a distinct survival signal to the cell and may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated.