脑缺血再灌注后bcl-2、caspase-3 mRNA水平表达与大脑皮质及纹状体区炎性细胞浸润

目的探讨大鼠脑缺血再灌注后bcl-2蛋白、caspase-3 mRNA的表达及炎性细胞浸润与神经细胞凋亡的关系。方法将54只Wistar大鼠随机分为二组：假手术组，缺血再灌注组。采用原位末端标记(TUNEL)、免疫组化和原位杂交技术分别观察脑缺血再灌注后不同时间点神经细胞凋亡及损伤的变化与bcl-2、caspase-3 mRNA表达。结果bcl-2表达于缺血再灌注12～24h达高峰，再灌注2—4d呈下降趋势，至16d略高于假手术组；caspase-3 mRNA于缺血再灌注12—24h达高峰，2—4d呈降低趋势，至16d略高于假手术组。结论脑缺血再灌注后细胞凋亡介导神经细胞损伤、坏死是一个渐进的动态演变过程。bcl-2蛋白、caspase-3 mRNA表达在抑制细胞凋亡和介导神经细胞损伤等方面起非常重要的作用。     

他克莫司通过减少肾小球足细胞凋亡降低2型糖尿病大鼠尿白蛋白

目的探讨他克莫司降低2型糖尿病(T2DM)大鼠尿白蛋白的可能机制。方法高脂高糖饲料喂养8周联合低剂量链脲佐菌素腹腔注射构建T2DM大鼠模型,随机选取后分为糖尿病模型组(DM组,n=10)和他克莫司(FK506)治疗组(FK组,n=10),另设正常对照组(n=10)。FK组给予FK506灌胃干预8周,DM组、正常对照组给予等量枸橼酸缓冲液灌胃8周。干预前后测定各组大鼠肾肥大指数(肾质量/体质量,KM/BM)、收缩压、24h尿白蛋白/尿肌酐,空腹血糖、内生肌酐清除率(CCr),总胆固醇、三酰甘油、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)和血白细胞;光镜、电镜观察肾脏病理改变;免疫组织化学方法测定nephrin蛋白表达;TUNEL法检测足细胞凋亡;Western blot法测定cleaved-caspase-3的表达和bax/bcl-2。结果与正常对照组相比,DM大鼠KM/BM、收缩压、空腹血糖、CCr、总胆固醇、三酰甘油及24h尿白蛋白/尿肌酐明显升高(均P0.05),经FK506干预后大鼠KM/BM、24h尿白蛋白/尿肌酐明显降低(均P0.05),而对收缩压、空腹血糖、CCr、ALT、AST和血白细胞的影响无统计学意义;光镜下,DM大鼠肾小球体积增大,系膜细胞增生,基底膜增厚;FK506干预后病理改变明显减轻;电镜下,DM组肾小球基底膜显著增厚,足突紊乱、融合率明显升高[(73.50±3.60)%比(4.20±1.91)%,P0.05];经FK506干预后上述病变有所减轻,足突融合率下降[(33.80±1.93)%比(73.50±3.60)%,P0.05];免疫组化结果显示,DM组肾组织nephrin蛋白表达较正常对照组下降,而FK506干预后nephrin蛋白表达明显恢复(P0.05);TUNEL法检测发现DM大鼠足细胞凋亡明显增多(P0.05),而FK506干预后足细胞凋亡明显减少(P0.05);Western blot结果显示:DM组肾组织cleaved-caspase-3蛋白表达和bax/bcl-2较正常对照组上升(P0.05),而FK组cleaved-caspase-3蛋白表达和bax/bcl-2明显降低(P0.05)。结论他克莫司可以通过减少糖尿病肾病大鼠足细胞的凋亡而发挥降低24h尿白蛋白,延缓肾功能减退的作用。     

灯盏花素对糖尿病大鼠肾脏细胞凋亡及bax、bcl-2表达的影响

目的探讨灯盏花素对糖尿病大鼠肾脏细胞凋亡及对bax和bcl-2 mRNA表达的影响。方法健康雄性SD大鼠随机分为正常对照组（NC）、糖尿病组（DM）和灯盏花治疗组（EB）,EB组行灯盏花素20mg·kg^-1·d^-1腹腔内注射。给药后2周和6周处死,检测肾脏细胞凋亡、肾脏bax和bcl-2 mRNA的表达。结果与NC组比较,（1）DM组及EB组肾小管凋亡细胞数明显增多,且DM组高于EB组;（2）DM组肾脏bax、bcl-2 mRNA的表达显著增强,bax／bcl-2值明显升高;EB组与DM组比较bcl-2 mRNA表达增强,bax mRNA减弱,bax／bcl-2比值降低;（3）EB组及DM组体重下降,肾指数、肾小球面积、体积升高,且EB组较DM组体重增加,肾指数、肾小球面积、体积下降。血尿素氮、肌酐比较：DM组〉EB组〉NC组。结论灯盏花素可能通过影响凋亡相关基因bcl-2和bax的表达而抑制。肾脏细胞凋亡,从而发挥肾脏保护作用。     

左卡尼汀强化St.Thomas No.2液对长时间冷保存离体心脏能量代谢的影响

目的 探讨不同剂量的左卡尼汀（LCN）添加至St.Thomas No.2液后对长时间冷保存离体大鼠心脏能量代谢的影响.方法 利用Langendorff灌注装置建立离体大鼠心脏模型.SD大鼠56只随机分为4组：对照组8只、St组16只（该组使用St.Thomas No.2液为心脏停搏液和保存液）、LCN 1组16只（St.Thomas No.2液加LCN 12g/L为心脏停搏液和保存液）和LCN 2组16只（St.Thomas No.2液加LCN 6g/L为心脏停搏液和保存液）.对照组直接取左心室心肌组织,其他各组16只大鼠中8只在4℃条件下保存心脏7h后取左心室心肌,另外8只建立离体左心工作模型,再灌注30min后取左心室心肌.检测心肌三磷酸腺苷（ATP）、二磷酸腺苷（ADP）、一磷酸腺苷（AMP）、总腺苷（TNA）含量并计算能荷（EC）;提取心肌线粒体后检测线粒体内Ca^2＋含量（[Ca^2＋]m）及线粒体呼吸功能.结果 St组冷保存后心肌ATP含量和EC显著下降,[Ca^2＋]m显著增高,线粒体呼吸控制率（RCR）明显降低（P〈0.05）;再灌注后心肌ATP含量及EC无明显改善,[Ca^2＋]m进一步增高,RCR进一步降低（P〈0.05）.LCN 1组及LCN 2组各项指标较St组显著改善（P〈0.05）,两治疗组之间比较差异亦有统计学意义.结论 不同剂量的左卡尼汀添加至St.Thomas No.2液可减轻线粒体钙超载,改善心肌能量代谢,对心肌细胞线粒体有较好的保护作用.此保护作用与LCN剂量有关.     

尼古丁对PD小鼠黑质多巴胺能神经元bcl-2、Fas蛋白表达的影响

目的观察尼古丁对帕金森病（PD）小鼠黑质神经元bcl-2、Fas蛋白表达影响。方法将28只小鼠随机分为正常对照组、尼古丁组、1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）组及尼古丁＋MPTP组各7只。MPTP组和尼古丁＋MPTP组均腹腔注射MPTP制作PD模型。尼古丁＋MPTP组制模前腹腔注射尼古丁;对照组腹腔注射等量生理盐水;尼古丁组仅腹腔注射尼古丁。末次给药后24 h黑质位置冠状切片,采用免疫组化法测定bcl-2、Fas蛋白表达情况。结果MPTP组bcl-2蛋白表达明显低于尼古丁＋MPTP组和对照组（P均〈0.01）;Fas蛋白表达MPTP组和尼古丁＋MPTP组明显高于对照组（P均〈0.01）,尼古丁＋MPTP组明显低于MPTP组（P〈0.01）。结论尼古丁可对抗MPTP所致bcl-2、Fas蛋白表达改变;本研究为PD的治疗提供了新思路。     

三氧化二砷诱导小细胞肺癌细胞凋亡及p53、bcl-2基因表达的研究

目的：研究三氧化二砷（As2O3）诱导小细胞肺癌细胞凋亡及其机制。方法：采用末端脱氧核苷酰转移（TUNEL）法检测细胞凋亡，免疫组织化学（SABC）法分析As2O3对小细胞肺癌细胞（NeI-H细胞）p53、bcl-2基因蛋白表达的影响。结果：As2O30.5μmol/L、1.0μmol/L、2.0μmol/L作用72h，NeI-H细胞均可呈现凋亡所特有的亚G1峰，凋亡比率随药物作用浓度增加和作用时间延长而增高。实验组p53基因蛋白表达较对照组明显增多，药物浓度越高表达越多（P=0.001）；bcl-2基因蛋白表达较对照组明显减少，药物作用浓度越高表达越少（P=0.001）。结论：As2O3抗肿瘤作用主要是通过诱导细胞凋亡实现的，其机制与上调p53基因表达及下调bcl-2基因表达有密切关系。     

bcl-2反义寡核苷酸与VP16协同抑制小细胞肺癌细胞增殖的研究

目的 研究bcl-2反义寡核苷酸与足叶乙甙（VPl6）联用对小细胞肺癌细胞NCI—H69增殖的影响。方法 通过脂质体将不同寡核苷酸导入NCI—H69细胞中，分为反义寡核苷酸组、正义寡核苷酸组、无义寡核苷酸组和空白对照组4组。Western Blot法检测细胞bcl-2蛋白的表达。不同浓度的VP16作用于4组转染后的细胞，MTT法测定细胞存活分数。结果 4组细胞中。与空白对照组相比较，反义寡核苷酸组的bcl-2蛋白表达明显受到抑制。bcl-2反义寡核苷酸转染细胞与VP16作用后，反义寡核苷酸组细胞在各个浓度的存活分数均低于对照组，且有统计学意义（P〈0．01）。结论 bcl-2反义寡核苷酸与VP16能协同抑制小细胞肺癌细胞的增殖。     

bcl-2在同型半胱氨酸致血管内皮细胞凋亡中的作用

目的:研究bcl-2在同型半胱氨酸致人脐静脉内皮细胞凋亡过程中的作用。方法:以人脐静脉内皮细胞株为实验对象,用含有不同浓度同型半胱氨酸的RPMI 1640培养液对其作用48h,MTT法测定细胞增殖生长能力;流式细胞术测定细胞凋亡水平;荧光定量PCR测定bcl-2基因的mRNA表达;Western blotting测定bcl-2蛋白表达量。结果:同型半胱氨酸对人脐静脉内皮细胞的增殖能力有明显的抑制作用,OD值从(0.99±0.05)渐下降至(0.28±0.03)(P0.01),且呈明显的剂量效应关系;细胞凋亡率由(2.05±0.26)%渐增至(30.86±1.94)%(P0.01);与对照组相比,实验组bcl-2mRNA及蛋白表达明显下降(P0.01)。结论:同型半胱氨酸可通过下调bcl-2的mRNA及蛋白质表达,促进人脐静脉内皮细胞的凋亡。     

SMO保存液对犬肾低温保存期间氧自由基变化的研究

建立犬离体肾脏单纯低温保存模型，对照组供肾灌注和保存用HTK保存液和UW保存液，实验组用自制SMO保存液，分别在犬肾保存24、48、72h切取肾皮质标本，测定皮质丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性。结果显示，实验组SOD活性48h明显高于对照组，MDA含量各时点均显著低于对照组（P均〈0．05）。实验组各时点SOD活性和MDA含量与UW液组比较均无统计学差异。提示SMO液在减轻氧自由基损伤方面优于HTK液，与UW液相当。     

原花青素对β-淀粉样肽（25—35）诱导PC12细胞bax和bcl-2基因表达的影响

目的探讨原花青素（Procyanidins，PC）对β-淀粉样肽（25-35）（hi325弗）诱导凋亡PCI2细胞bax和bcl-2基因表达的影响。方法采用MTT比色法分析细胞存活率，Hoechst33258-PI荧光染色观察细胞核凋亡的形态学改变，RT—PCR检测bax和bcl-2基因mRNA表达，Western blot检测Bax和Bcl-2蛋白表达。结果不同剂量PC预处理PC12细胞1h，可剂量依赖性对抗Aβ25-35引起的细胞凋亡，增加细胞存活率，减少Aβ25-35引起的细胞核固缩、核碎裂，降低baxmRNA及Bax蛋白表达，增加bcl-2mRNA及Bcl-2蛋白表达。结论PC可剂量依赖性对抗Aβ25-35诱导的PCI2细胞凋亡，其机制可能与下调凋亡基因bax和上调抗凋亡基因bcl-2表达有关。     

Variants of bcl-2 specific siRNA for silencing antiapoptotic bcl-2 in pancreatic cancer

BACKGROUND AND AIMS: Pancreatic cancer remains a devastating diagnosis with only limited therapeutic options. Specific inhibition of expression of target genes has become possible using small interfering (si) RNAs. We therefore investigated how far siRNA specific for bcl-2 may serve as a therapeutic option for pancreatic cancer in vitro and in vivo. METHODS: siRNAs targeting two different regions in the bcl-2 gene were transfected to YAP C and DAN G pancreatic carcinoma cells and human foreskin fibroblasts. Permutations were generated by changing 3' and 5' overhangs and varying the length of the paired RNA duplex. Transfection efficacy was determined using FITC labelled siRNAs and fluorescence microscopy. Cell survival and apoptosis were quantified at 24-120 hours. Pancreatic cancer xenografts in male nude mice were treated intraperitoneally with siRNAs daily for 24 days. siRNA pharmacokinetics in vivo were assessed using radioactively labelled siRNAs. Total protein and RNA were extracted for western Blot analysis and quantitative polymerase chain reaction. RESULTS AND CONCLUSIONS: Bcl-2 specific siRNAs specifically inhibited expression of the target gene in vitro and in vivo. Antiproliferative and proapoptotic effects were observed in tumour cells but not in fibroblasts or non-malignant tissues. siRNA permutations and diverse overhangs influenced gene silencing efficacy. siRNA was quickly distributed to all organs and excreted via the kidney and liver. Bcl-2 specific siRNA is a promising adjunctive treatment for pancreatic carcinoma.     

bcl-2 expression in non-Hodgkin's lymphomas is not associated with bcl-2 gene rearrangements

Previous reports have associated bcl-2 gene rearrangements found in non-Hodgkin's lymphomas with an inappropriately elevated bcl-2 expression compared with the mature B-cell stage of development. This study investigates bcl-2 expression in non-Hodgkin's lymphomas (NHL) without bcl-2 gene rearrangements. Molecular analysis in 168 patients with NHL revealed 45 patients without bcl-2 gene rearrangements in which additional immunostaining for bcl-2 protein was possible. An unexpectedly high prevalence (39/45) of bcl-2 expression was found. The levels and patterns of bcl-2 expression were not specific for the histological type of NHL and were similar to those shown in comparable cases with bcl-2 gene rearrangements. In conclusion, bcl-2 expression is not specific for NHL bearing bcl-2 gene rearrangements. This finding implicates the existence of other deregulating control mechanisms of bcl-2 expression, more important than bcl-2 gene rearrangements.     

B-CLL and bcl-2 gene

Most of human follicular lymphomas (approximately 90% in U.S.A. or approximately 30% in Japan) possess the t(14; 18) chromosome translocation that directly involves the IgH locus on chromosome 14 and the bcl-2 gene on chromosome 18. The t(14; 18) chromosome translocation occurs nearly exclusively at two hot spots, a major breakpoint clustering region (mbr) within the 2nd exon noncoding region and the minor breakpoint clustering region (mcr) within the 3' flanking region of the bcl-2 gene. Here we show that the rearrangement of the bcl-2 gene occurs in a significant fraction (approximately 10%) of B-CLL. All of the rearranged bcl-2 genes were juxtaposed with Ig lambda or Ig kappa genes, implying that the bcl-2 gene is preferentially linked to the IgL genes in CLL.     

Detection of bcl-2 and bax expression and bcl-2／JH fusion gene in intrahepatic cholangiocarcinoma

AIM: To investigate the relationship between bcl-2 gene and its related protein bax and intrahepatic cholangiocellular carcinoma (CCC). METHODS: Semi-nested in situ PCR (SNISPCR) and immunohistochemistry were performed to detect bcl-2/JH fusion gene and bcl-2, bax protein expression in 29 cases of CCC. RESULTS: No bcl-2/JH fusion gene was found in all cases of CCC, 72.4% of 29 cases expressed bcl-2 protein. Bcl-2 protein expression was related to histopathological grades (P<0.05). There was no corresponding relationship between bcl-2/JH fusion gene formation and bcl-2 protein expression in CCC (P<0.05). Bax was expressed in 10.3% of 29 cases. The ratio of bcl-2 to bax in normal liver tissues (3.5 to 1) was different from that in tumor tissues (7.0 to 1). CONCLUSION: It is suggested that bcl-2/JH fusion gene formation is not a frequent event and may not play an important role in the pathogenesis of CCC. However, aberrant ratio of bcl-2 to bax protein expression may be involved in the course of tumorigenesis of CCC. Abnormal bcl-2 protein expression may not be solely resulted from bcl-2/JH fusion gene.     

bcl-2 overexpression promotes myocyte proliferation

To determine the influence of Bcl-2 on the developmental biology of myocytes, we analyzed the population dynamics of this cell type in the heart of transgenic (TG) mice overexpressing Bcl-2 under the control of the alpha-myosin heavy chain promoter. TG mice and non-TG (wild type, WT) mice were studied at 24 days, 2 months, and 4 months after birth. Bcl-2 overexpression produced a significant increase in the percentage of cycling myocytes and their mitotic index. These effects were strictly connected to the expression of the transgene, as demonstrated in isolated myocytes. The formation of mitotic spindle and contractile ring was identified in replicating cells. These typical aspects of mitosis were complemented with the demonstration of karyokinesis and cytokinesis to provide structural evidence of cell division. Apoptosis was low at all ages and was not affected by Bcl-2. The higher cell replication rate in TG was conditioned by a decrease in the expression of the cell-cycle inhibitors, p21(WAF1) and p16(INK4a), and by an increase in Mdm2-p53 complexes. In comparison with WT, TG had 0.4 x 10(6), 0.74 x 10(6), and 1.2 x 10(6) more myocytes in the left ventricle at 24 days, 2 months, and 4 months, respectively. Binucleated myocytes were 12% and 25% larger in WT than in TG mice at 2 and 4 months of age. Taken together, these observations reveal a previously uncharacterized replication-enhancing function of Bcl-2 in myocytes in vivo in the absence of stressful conditions.     

小儿急性白血病bcl—2、bax蛋白的表达及临床意义

目的：检测小儿急性白血病bcl-2、bax蛋白的表达，探讨其与临床诊治、预后的关系及两基因间的相互作用。方法：采用免疫细胞化学技术分析患儿骨髓单个核细胞bcl-2、bax蛋白的表达。结果：初治组bcl-2蛋白表达较CR一和正常组显著增高；bax表达显著降低；bcl－2表达与影响预后因素间无显著相关。结论：bcl-2抑调亡作用增强和bax促凋亡作用减弱在小儿白血病的发生发展中具有重要作用，bcl－2表达可能与疗有关，bcl-2、bax共同调控白血病细胞凋亡。     

EXPRESSION OF bcl-2 IN RHEUMATOID ARTHRITIS

Since defective apoptosis has been suggested to play a rolein the development of autoimmune diseases, we have investigatedthe expression of the proto-oncogene bcl-2 in patients withrheumatoid arthritis (RA). The expression of bcl-2 was studiedin peripheral blood (PB) and synovial fluid (SF) lymphocytesand synovial tissues (ST) from patients with RA using immunohistochemistry,flow cytometry and nucleic acid hybridization. Patients withreactive arthritis (ReA) or osteoarthritis (OA) and healthyindividuals were used as controls. The expression of bcl-2 proteinin PB lymphocytes and the expression of bcl-2 mRNA in PB mononuclearcells (PBMC) was similar in healthy controls and patients withRA. However, bcl-2 protein expression was significantly reducedin SF lymphocytes when compared to PB lymphocytes. Similar resultswere observed with lymphocytes from patients with ReA, and irrespectiveof whether total lymphocytes, T cells or different T-cell subsetswere studied. In the synovial sections, the expression of bcl-2was restricted to lymphocytes, and bcl-2+ cells were observedin the majority of samples from patients with RA, OA and ReA.These data indicate that the expression of bcl-2 is not increasedin the lymphocytes or ST derived from patients with RA. Instead,decreased expression of bcl-2 protein in SF lymphocytes comparedto PB lymphocytes was demonstrated. We suggest that bcl-2 doesnot play a significant role in the pathogenesis of RA. KEY WORDS: bcl-2, Rheumatoid athritis, Lymphocytes     

bcl-2 gene in B cell lymphoma

In t(14;18) lymphomas, bcl-2 gene is activated by the juxtaposition of immunoglobulin (Ig) gene. The fused bcl-2-Ig gene generates chimeric mRNAs which consist of bcl-2 at 5' side and Ig at 3' side. Chimeric mRNA does not disrupt the bcl-2 coding frame of 239 amino acid polypeptide. Activated bcl-2 gene introduced in normal B lymphoblastoid cells (LCL) demonstrated an increased cloning efficiency in soft agar but failed to confer tumorigenicity to LCLs as a single agent. bcl-2 gene rearrangement in Japaneses B cell lymphoma was studied and found that 10 out of 32 cases of follicular lymphoma (31%) and 5 out of 56 cases of diffuse lymphoma (9%) were rearranged, suggesting less frequency of B cell lymphoma, particularly follicular lymphoma in Japan is partly due to less bcl-2 involvement than American cases. Three cases out of 15 cases with bcl-2 rearrangement demonstrated a unique pattern of rearrangement. Two cases of the three were analysed and found that both cases were translocated at the later step than DH-JH joining of Ig rearrangement. Thus, bcl-2 translocation in Japanese B cell lymphomas might occur at the later stage of B cell development, when compared with that in American cases. Less involvement of bcl-2 in Japanese B cell lymphoma may be explained by low susceptibility to bcl-2 rearrangement at the step of DH-JH recombination.     

Solution structure of the antiapoptotic protein bcl-2

The structures of two isoforms of Bcl-2 that differ by two amino acids have been determined by NMR spectroscopy. Because wild-type Bcl-2 behaved poorly in solution, the structures were determined by using Bcl-2/Bcl-x(L) chimeras in which part of the putative unstructured loop of Bcl-2 was replaced with a shortened loop from Bcl-x(L). These chimeric proteins have a low pI compared with the wild-type protein and are soluble. The structures of the two Bcl-2 isoforms consist of 6 alpha-helices with a hydrophobic groove on the surface similar to that observed for the homologous protein, Bcl-x(L). Comparison of the Bcl-2 structures to that of Bcl-x(L) shows that although the overall fold is the same, there are differences in the structural topology and electrostatic potential of the binding groove. Although the structures of the two isoforms of Bcl-2 are virtually identical, differences were observed in the ability of the proteins to bind to a 25-residue peptide from the proapoptotic Bad protein and a 16-residue peptide from the proapoptotic Bak protein. These results suggest that there are subtle differences in the hydrophobic binding groove in Bcl-2 that may translate into differences in antiapoptotic activity for the two isoforms.