BAD和Bcl-X/L基因在小鼠胸腺细胞凋亡时的表达及其意义

目的检测BAD和Bcl-X/L基因在小鼠胸腺细胞凋亡的表达,探讨细胞凋亡调控基因在小鼠胸腺细胞凋亡时的作用。方法应用免疫组织化学染色法,观察不同剂量糖皮质激素诱导的小鼠胸腺细胞凋亡时BAD和Bcl-X/L基因的表达。结果免疫组织化学结果表明,正常对照组胸腺内BAD呈低表达,地塞米松组随剂量的增加BAD的表达成递增趋势,地塞米松各组与对照组比较差异有有统计学意义(P<0.05);正常对照组胸腺内Bcl-X/L呈高表达,地塞米松组随剂量的增加Bcl-X/L的表达成递减趋势,地塞米松各组与对照组比较差异有统计学意义(P<0.05)。结论促凋亡蛋白BAD和抗凋亡蛋白Bcl-X/L在糖皮质激素诱导引起的胸腺细胞凋亡调控中起重要作用。     

食管上皮癌变过程中FAS、FASL、FADD和caspase-8的表达及其意义

目的研究细胞凋亡调控因子FAS、FASL、FADD和caspase-8基因在食管上皮癌变过程中的表达及其意义。方法用免疫组化SP法检测食管黏膜中FAS、FASL、FADD和caspase-8的表达;原位杂交法检测食管黏膜中FADDmRNA和caspase-8mRNA的表达。结果从食管正常黏膜组织到不典型增生和食管癌组织,FAS蛋白表达率呈逐渐下降趋势,FASL蛋白表达率呈逐渐上升趋势,食管癌与正常组织之间差异明显(P<0·05)。高、中分化鳞癌中蛋白阳性表达率显著高于低分化鳞癌(P<0·05)。从食管正常黏膜组织到不典型增生和食管癌组织,FADD和caspase-8蛋白及RNA阳性表达率呈逐渐下降趋势,食管癌与正常黏膜组织之间差异明显(P<0·01);caspase-8蛋白和mRNA在高、中分化鳞癌中的表达率高于低分化鳞癌(P<0·05)。结论FAS与FASL的表达失衡与食管癌的发生、发展有密切关系,并与食管癌的分化和免疫逃避有关。FADD与caspase-8基因表达异常在食管癌的发生、发展中可能起着重要作用。     

Protection of growth hormone on apoptosis of murine thymocytes induced by dexamethasone

目的:研究生长激素(GH)对地塞米松(Dex)诱导小鼠胸腺细胞凋亡的作用及机制.方法:建立地塞米松诱导小鼠胸腺细胞凋亡模型,通过观察小鼠胸腺指数的变化、形态学改变,TdT缺口末端标记(TUNEL)及流式细胞仪检测,研究生长激素影响胸腺细胞凋亡情况,并运用免疫组织化学观察生长激素对小鼠胸腺凋亡蛋白Bax和Bcl-2表达的调节.结果:经生长激素处理后,电镜下观察小鼠胸腺细胞凋亡现象明显改善;TUNEL检测和流式细胞仪检测结果显示,胸腺细胞凋亡数量减少;图像分析系统测定光密度值显示,生长激素处理组小鼠胸腺细胞中Bax表达较模型组降低,Bcl-2表达增加.结论:生长激素对地塞米松诱导的小鼠胸腺细胞凋亡具有一定的抑制作用,这种作用可以通过调节凋亡蛋白Bax、Bcl-2的表达等途径实现.     

糖皮质激素诱导小鼠胸腺细胞死亡的形态学改变

目的 探讨糖皮质激素诱导小鼠胸腺细胞死亡的形态学改变. 方法 4～6周龄雌性BALB/C小鼠腹腔注射地塞米松,采用末端转移酶介导的dUTP缺口末端标记(TUNEL)法检测DNA单链或双链的裂解,TUNEL和酸性磷酸酶(ACP)双重染色法进行酸性磷酸酶的检测,观察细胞凋亡后吞噬细胞的活动,透射电子显微镜检测凋亡细胞和吞噬细胞的超微结构.结果 光镜观察地塞米松组,TUNEL阳性细胞较多并且分散在皮质各处,凋亡指数为0.460±0.012;正常对照组TUNEL阳性细胞数目较少,凋亡指数为0.020±0.001,与地塞米松组比较,差异有显著性(P<0.05).TUNEL和酸性磷酸酶双重染色法显示,所有TUNEL阳性胸腺细胞均被ACP阳性细胞吞噬.透射电子显微镜观察发现,大部分凋亡细胞均被吞噬细胞所吞噬,少量未被吞噬的胸腺细胞表现出大范围的染色质浓集,这些少量未被吞噬的胸腺细胞显然是死细胞. 结论 典型的细胞凋亡的特点是DNA裂解,但这不是糖皮质激素诱导胸腺细胞死亡的主要方式,死亡的胸腺细胞均是被ACP阳性细胞所吞噬;糖皮质激素对胸腺细胞的首要影响是可诱导无DNA裂解的细胞固缩.     

雌激素对大鼠胸腺细胞凋亡及Bcl-2、Bax表达的影响

目的:探讨苯甲酸雌二醇对大鼠胸腺Bcl-2和Bax表达及细胞凋亡的影响及其机制.方法:雌性大鼠行卵巢切除术,给予苯甲酸雌二醇后,观察胸腺指数的变化,Hochest33342荧光染色及透射电镜标本观察胸腺细胞凋亡情况,免疫组织化学检测胸腺组织中Bcl-2和Bax的表达情况,原位杂交技术检测Bcl-2、Bax m RNA的表达情况.结果:双侧卵巢切除组大鼠胸腺指数较假手术组增加,双侧卵巢切除+雌激素组大鼠胸腺指数较双侧卵巢切除组减小;假手术组和双侧卵巢切除组大鼠胸腺组织中以正常胸腺细胞为主,偶见凋亡细胞或凋亡小体,双侧卵巢切除+雌激素组可见较多凋亡细胞和凋亡小体;双侧卵巢切除+雌激素组大鼠胸腺组织中Bcl-2表达较双侧卵巢切除组和假手术组增高明显降低,而Bax表达呈现相反趋势;Bcl-2 mRNA、Bax mRNA的表达与Bcl-2、Bax的表达呈一致性.结论:雌激素可以降低大鼠胸腺指数,抑制胸腺组织中Bcl-2的表达,促进Bax的表达,从而诱导大鼠胸腺细胞凋亡,促进雌性大鼠胸腺退化.     

凋亡诱导因子在慢性砷中毒大鼠附睾上皮细胞凋亡中的表达

目的:探讨砷对大鼠附睾上皮细胞凋亡及凋亡诱导因子(AIF)和核酸内切酶G蛋白表达的影响。方法:雄性SD大鼠随机分为对照组和亚砷酸钠染毒组。采用自由饮用方式进行连续染毒6个月。采用TUNEL细胞凋亡染色法测定大鼠附睾上皮凋亡细胞的凋亡情况,采用免疫组织化学测定附睾组织内AIF、核酸内切酶G的分布,蛋白免疫印迹检测AIF、核酸内切酶G及具有活性的已剪切的cl-caspase-3及cl-caspase-9的表达水平。结果:免疫组织化学结果显示,与对照组比较,砷中毒组大鼠附睾内AIF、核酸内切酶G蛋白阳性表达明显升高,差异有统计学意义;TUNEL法结果显示,与对照组比较,砷中毒组大鼠附睾的细胞凋亡水平较高,差异均有统计学意义;蛋白免疫印迹结果显示与对照组比较,砷中毒组大鼠附睾内AIF、核酸内切酶G、cl-caspase-3及cl-caspase-9蛋白表达水平均较高,差异有统计学意义。结论:慢性砷中毒时以AIF为主的非caspase依赖性凋亡途径和caspase介导的经典途径共同参与了大鼠附睾组织的损伤及凋亡。     

缺血预适应对大鼠心肌细胞凋亡及bcl-2、bax蛋白表达的影响

目的：探讨缺血预适应对大鼠心肌细胞凋亡及bcl-2、bax蛋白表达的影响。方法：以DNA电泳和TUNEL分析检测大鼠心肌组织的细胞凋亡情况，免疫组织化学方法检测bcl-2、bax蛋白的表达。结果：缺血再灌注组缺血区心肌DNA电泳呈现典型的DNA梯形，而缺血预适应组和假手术对照组未见梯形。缺血预适应组心肌细胞凋亡指数显著低于缺血再灌注组（P<0.01）。Bcl-2在各组均无阳性表达，bax在各组表达无显著差异（P>0.05）。结论：缺血预适应显著减少了缺血再灌注诱导的心肌细胞凋亡程度。     

γ辐射诱发的人AHH-1 T淋巴细胞凋亡及其调控机制

目的：研究电离辐射诱发正常人AHH-1 T淋巴母细胞凋亡的特点及其调控机制，为辐射免疫损伤的防治提供实验依据。方法：用TUNEL、麦格-姬姆萨(MGG)法、MTT比色法及碱磷酶免疫组织化学染色法。分别检测T细胞凋亡、细胞活存以及Bax、Bcl-XL和caspase-3重要凋亡相关蛋白的表达及规律。结果：①在一定照射剂量范围内随照射剂量的增加，AHH-1 T细胞凋亡率持续升高，而细胞活的活存率则急剧下降，两者均呈现明显的剂量效应关系。②促凋亡蛋白Bax的表达随照射剂量的增加明显增强，而凋亡抑制蛋白Bcl-XL则呈持续下降的趋势。③活化的凋亡执行蛋白caspase-3的活性随照射剂量的增加显著升高，与凋亡率呈现明显的相应关系。结论：AHH-1 T细胞的大量凋亡，可能是辐射导致淋巴细胞急剧减少、机体免疫功能降低的重要原因；Bax、Bcl-XL和caspase-3蛋白在辐射诱发的T细胞凋亡调控中具有重要作用。     

中药六味地黄汤对衰老大鼠卵巢组织Bax/Bcl-2及Caspese-3蛋白表达的影响

目的探讨六味地黄汤对衰老大鼠卵巢组织凋亡相关基因Bax/Bcl-2及Caspese-3蛋白表达的影响。方法D-半乳糖连续腹腔注射致亚急性衰老动物模型,造模后灌胃何首乌饮连续60天后,大鼠断头取卵巢。采用免疫组织化学分析方法检测各组大鼠凋亡相关基因Bcl-2,Bax的表达;Western Bloting法检测各组大鼠卵巢Caspese-3蛋白表达的变化。结果模型组较正常组卵巢细胞Bax、Caspse-3表达量增加,Bcl-2表达减弱;六味地黄汤可使卵巢Bax、Caspse-3表达量降低,Bcl-2表达增加。差异有统计学意义(P<0.05)。结论六味地黄汤剂能延缓衰老大鼠卵巢细胞凋亡的发生,其机制可能是通过干预凋亡相关基因Bax/Bcl-2及Caspese-3蛋白表达抑制卵巢细胞的凋亡。     

抗BrdU单克隆抗体在凋亡细胞检测中的应用研究

目的 探讨将自制的抗BrdU单克隆抗体（mAb）用于TUNEL法检测凋亡细胞的可能性。方法 分别用TUNEL免疫荧光法和免疫组化技术，检测地塞米松诱导的凋亡胸腺细胞和射线照射的大鼠皮肤组织中的凋亡细胞。结果 用TUNEL免疫荧光法和TUNEL免疫且化法法检测发现，凋亡细胞的细胞核分别呈明显的荧光或酶底物着色，而非凋亡细胞不显示荧光或无酶底物着色。结论 该抗BrdU mAb用于TUNEL法，能够检测单个细胞标本或组织切片中的凋亡细胞，为国内开展凋亡细胞的检测，提供了一种新的方法。     

Characteristic of readiness of thymocytes and peripheral blood lymphocytes to proliferation and apoptosis in experimental chronic liver disease

Indices of of thymocyte and peripheral blood lymphocyte readiness to apoptosis and proliferation were studied in experimental chronic liver disease of toxic and autoimmune genesis. Smears of thymocytes and peripheral blood lymphocytes were examined. Readiness to programmed cell death was determined by indirect immunofluorescence reaction with monoclonal antibodies to detect FAS/APO antigen (CD95), known to mediate apoptosis. Proliferative activity was estimated by counting nucleolar organizing regions. Indices of thymocyte readiness to apoptosis and proliferation were found to decrease in experimental animals, while similar parameters of peripheral blood lymphocytes increased. These data suggest the increased sensitivity of immunocompetent cells to activating signals, which might result in impaired immune response.     

Exercise training enhances tumor necrosis factor-alpha-induced expressions of anti-apoptotic genes without alterations in caspase-3 activity in rat epididymal adipocytes

The effect of exercise training on tumor necrosis factor-alpha (TNF-alpha) signaling was investigated in rat epididymal adipocytes. Incubation of isolated adipocytes with TNF-alpha (20 ng/ml) for 5 h enhanced the expression of the inhibitor apoptosis protein 2 (IAP2) gene without any enhancement of caspase-3 activity in both the sedentary control (C) and exercise-trained (TR) groups. However, the ability of TNF-alpha to enhance IAP2 gene expression was significantly greater in TR than in C rats. The basal expression of the IkappaB kinase beta (IKK beta) gene and that of the BCL-x(L) gene were also higher in TR than in C rats. Mn-superoxidedismutase contents in adipocytes were higher in TR than in C rats. Moreover, no apoptotic nucleuses of adipocytes in response to acute exercise were observed in either group at least up to 5 h after exercise. Exercise training also enhanced the inhibitory effect of TNF-alpha on the gene expression of the fatty acid synthase (FAS), a lipogenic enzyme, suggesting that fatty acid synthesis may be reduced. Thus, exercise training enhanced TNF-alpha signaling directed toward the expressions of survival signals and the suppression of FAS gene expression.     

Gene expression analysis of thymocyte selection in vivo

Self versus non-self discrimination is a key feature of immunorecognition. Through TCR-activated apoptotic mechanisms, autoreactive thymocytes are purged at the CD4(+)CD8(+) double-positive (DP) precursor stage prior to maturation to CD4(+) or CD8(+) single-positive (SP) thymocytes. To investigate this selection process in vivo, gene expression analysis by oligonucleotide array was performed in TCR transgenic mice. In total, 244 differentially expressed DP thymocyte genes induced or repressed by TCR triggering in vivo were identified. Genes involved in the biological processes of apoptosis, DNA recombination, antigen processing and adhesion are coordinately engaged. Moreover, analysis of gene expression in thymocyte subsets revealed that TCR ligand-induced expression profiles vary according to their developmental stage, with 48 genes showing DP preference and nine showing SP thymocyte preference. Finally, our data suggest that both the extrinsic and the intrinsic apoptosis pathways are operating in thymic selection.     

白介素10对脑缺血大鼠神经细胞凋亡的作用研究

目的:探讨白介素10(IL-10)对大鼠脑缺血梗死灶周围神经细胞凋亡的作用。方法:成年雄性Sprague-Darley大鼠36只,随机分为假手术组(Sham组)、局灶性脑缺血组(MCAO组)和脑缺血 IL-10干预组(IL-10组),术后24h断头取脑,TUNEL法(Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling)测定梗死灶周围凋亡神经细胞的数目,免疫组化和RT-PCR法检测促凋亡基因Fas、FasL和caspase-3的表达。结果:脑缺血诱导神经细胞凋亡显著增多(P<0.05),Fas,FasL和caspase-3表达显著上调(P<0.05);IL-10干预可显著减少脑缺血神经细胞凋亡(P<0.05),并抑制FasL和caspase-3的表达(P<0.05),而对Fas的表达无明显作用(P>0.05)。结论:IL-10可抑制大鼠脑缺血梗死灶周围神经细胞凋亡,其机制可能与抑制促凋亡基因FasL和caspase-3的表达有关。     

RNA interference-mediated silencing of the p53 tumor-suppressor protein drastically increases apoptosis after inhibition of endogenous fatty acid metabolism in breast cancer cells

Fatty acid synthase (FAS)-dependent endogenous fatty acid synthetic activity is abnormally elevated in a biologically-aggressive subset of breast carcinomas. Remarkably, tumor-associated FAS hyperactivity represents a novel target for anti-metabolic therapy because pharmacological inhibitors of FAS are selectively cytotoxic for tumor cells, triggering their apoptotic cell death. Since the p53 tumor-suppressor protein (TP53) is thought to play a novel role in cellular responses of a variety of non-genotoxic metabolic stresses, we characterized the involvement of TP53 in the response of breast cancer cells to FAS inhibition. MCF-7 breast cancer cells were selected for study because they have an intact TP53 pathway and undergo little apoptosis following FAS blockade. Two chemically distinct inhibitors of FAS (the natural mycotoxin cerulenin and the novel small-molecule inhibitor C75) were studied in parallel to provide a broad picture of consequences suffered by the loss of FAS function on TP53 signaling. Treatment with either cerulenin or C75 induced TP53 protein accumulation at 24 h in MCF-7 cells. To determine whether the up-regulation of TP53 following exposure to cerulenin or C75 was solely due to inhibition of endogenous fatty acid metabolism, we first evaluated the cytotoxic response to chemical FAS blockers on MCF-7 cells in which FAS gene expression was previously silenced by using the highly sequence-specific mechanism of RNA interference. MCF-7 cells became insensitive to C75-induced cytotoxicity when the expression of FAS was specifically suppressed by targeted knock-down with small interfering RNA (siRNA), whereas they partially retained their sensitivity to cerulenin. These results demonstrate that C75-induced cytotoxic damage to breast cancer cells is closely dependent on its ability to inhibit FAS-catalyzed endogenous fatty acid biogenesis, thus ruling out a significant direct effect of C75 on DNA. To determine the functional role of TP53 on breast cancer cell survival after FAS blockade, we evaluated FAS inhibitor-mediated apoptosis in MCF-7 cells transiently transfected with a pool of sequence-specific double-stranded RNA oligonucleotides targeting TP53 gene. In these conditions, TP53 protein levels were unchanged during the period of FAS-inhibitor exposure. Remarkably, siRNA-induced silencing of TP53 gene expression did result in a dramatic increase (approximately 300%) in apoptotic cell death following exposure to C75. Strikingly, there was no apparent relationship between the TP53 mutational status and sensitivity to chemical FAS inhibitor in a panel of human breast cancer cell lines. However, the degree of TP53 mRNA expression was predictive of sensitivity to C75-induced cytotoxicity, with low-TP53 mRNA expressing breast cancer cells showing hypersensitivity to FAS blockade. These findings strongly suggest that: a) TP53 is a novel molecular sensor of energy imbalance after the perturbation of endogenous fatty acid metabolism in breast cancer cells; b) TP53 function closely influences the decision between apoptosis and growth arrest following FAS blockade; and c) pharmacological inhibitors of FAS activity may be clinically useful against breast carcinomas exhibiting mutation or aberrant expression of TP53.     

孕激素对更年期雌性大鼠脾细胞凋亡及相关基因的调控作用

为了解孕激素对更年期雌性大鼠脾细胞凋亡及相关基因的调控作用。首先通过应用Annexin V-FITC/PI双染色流式细胞仪测定脾细胞凋亡率;通过RT-PCR检测脾细胞凋亡基因Bcl-2、Bax的表达;RIA法测血孕酮(P)水平。结果发现更年期组血孕激素水平明显低于青年组和孕激素组(P<0.01),孕激素组与青年组相比差别无显著意义(P>0.05);更年期组大鼠脾细胞凋亡率高于青年组(10.3%和7.7%,P<0.05),促凋亡基因Bax的转录水平也高于青年组;孕激素组脾细胞的凋亡率与更年期组比较无显著差异,孕激素组促凋亡的Bax基因转录水平高于更年期组大鼠(P<0.01),而Bcl-2基因未见显著差异。研究结果提示,衰老过程中脾淋巴细胞凋亡增加,并受到相关基因的调控,孕激素治疗未能逆转更年期雌性大鼠脾细胞凋亡的增加,且使更年期大鼠脾细胞促凋亡的Bax基因表达上调。     

Developmental regulation of bcl-2 expression in the thymus.

An important factor in shaping the T-cell receptor (TcR) repertoire during thymocyte development is the susceptibility of double-positive (CD4+ CD8+) thymocytes to induction of apoptosis (negative selection) when the TcR is engaged by 'self'-antigens. Recent evidence has suggested that this susceptibility to apoptosis may be influenced by the expression of bcl-2, a proto-oncogene known to increase the resistance to apoptosis in various cell systems. Using a semi-quantitative polymerase chain reaction (PCR) technique in conjunction with staged embryonic material and purified thymocyte subpopulations we have investigated patterns of bcl-2 expression during normal T-cell development. Our results show that while bcl-2 alpha gene expression is readily detectable in immature CD3-CD4-CD8- thymocytes and in mature single-positive TcRhi cells, it is drastically reduced in TcR negative double-positive (CD3- CD4+ CD8+) cortical thymocytes of intermediate maturity. Careful mapping of bcl-2 alpha re-expression in relation to the onset of TcR expression within the population of embryonic thymocytes indicates that bcl-2 alpha is up-regulated as soon as TcR molecules are expressed on the surface of CD4+ CD8+ thymocytes. Therefore, thymocytes susceptible to apoptosis on TcR ligation express bcl-2 alpha mRNA suggesting that changing levels of bcl-2 expression are unlikely to be the only determinant regulating susceptibility to apoptosis in the thymus. The possible implications of these changes in bcl-2 expression regarding other facets of thymocyte development will be discussed.     

FAS and FAS-L expression by tumor cells and lymphocytes in breast carcinomas and their lymph node metastases

FAS receptor (FAS, CD95) and FAS ligand (FAS-L, CD95-L) are complementary members of a particular apoptotic pathway that plays a major role in immune regulation. The activation of FAS-L may trigger cytotoxic mechanisms leading to the death of FAS-expressing cells. Tumor cells and tumor-infiltrating lymphocytes (TIL) may express FAS and FAS-L in various proportions, and their interplay may affect tumor behavior. In the present study, we explored the expression of FAS and FAS-L in 28 mammary carcinomas (19 ductal and 9 lobular) and in their lymph node metastases. The expression of these mediators in immunostained sections was graded and evaluated comparatively between normal and neoplastic mammary epithelium, between tumor cells and TILs, and between mammary carcinoma cells and their lymph node metastases. We demonstrated the coexpression of FAS and FAS-L by breast carcinoma cells and TIL, with FAS expressed more strongly by normal epithelial cells and TIL than tumor cells. FAS-L was better stained on tumor cells than on TIL. There was equal or greater expression of FAS and FAS-L in the primary tumors and their TIL than in the metastatic counterparts. Comparing the expression of FAS with that of FAS-L, we recorded FAS equal or stronger than FAS-L in the primary mammary tumors and the reversal of their expression, FAS-L greater than FAS in the lymph node metastases. These results are consistent with reports of studies with other tumors, suggesting that the upregulated FAS-L indicates an increased ability of tumor cells to induce apoptosis in TIL and in the normal tissues invaded. However, it is understood that the FAS/FAS-L system, although essential for apoptosis, is only a contributing factor to the complex process of tumor invasion and antitumor defense.