pTAT-XIAP重组表达载体的构建与酶切鉴定

目的 探索pTAT-XIAP融合表达载体的构建方法.方法 在体外合成大鼠XIAP基因,将其插入pTAT-HA质粒,构建pTAT-HA/XIAP融合表达载体,将pTAT-HA/XIAP质粒转化至DH5α感受态细胞, 筛选培养转化成功的单克隆,NcoI/XhoI双酶切后琼脂糖电泳鉴定.结果 转化pTAT-HA/XIAP质粒的DH5α在含氨苄青霉素的LB固体培养基上呈分散菌落生长.电泳结果显示重组pTAT-HA/XIAP质粒约4 500 bp,酶切后可观察到PTAT-HA质粒约3 000 bp,XIAP目的 基因条带1 494 bp,pTAT空质粒约3 000 bp,条带大小与质粒图谱一致.结论 将大鼠XIAP基因成功插入pTAT-HA质粒,构建pTAT-XIAP融合蛋白表达载体,提取的质粒可用于下游分子生物学实验.     

XIAP蛋白在非霍奇金淋巴瘤中的表达及其临床病理意义

目的探讨XIAP蛋白在非霍奇金淋巴瘤（NHL）中的表达及其临床病理意义。方法应用TdT介导的dUTP缺口末端标记（TUNEL）技术和免疫组织化学SP法,检测79例NHL和20例良性淋巴结病变中的肿瘤细胞凋亡及XIAP蛋白的表达水平。结果39．2％（31／79）NHL表达XIAP蛋白,而在15％（3／20）良性淋巴结病变中弱阳性表达,两者差异有统计学意义（P〈0．05）;XIAP在NHL中的表达与年龄、性别及免疫表型无关（P〉0．05）：XIAP阳性表达患者的平均生存时间明显短于无表达患者（P〈0．05）。结论XIAP蛋白可能通过凋亡抑制功能在NHL的发生进展中有一定作用,并可作为判断NHL预后的参考指标。     

重组人红细胞生成素对癫痫持续状态大鼠海马p-Akt和XIAP的影响

目的 观察重组人红细胞生成素(recombinant human EPO,rHuEPO)对戊四氮(Pentylenetetrazol,PTZ)点燃的癫痫持续状态(status epilepticus,SE)的SD大鼠海马神经元磷酸化蛋白激酶B(p-PKB/p-Akt)和X-连锁凋亡抑制蛋白(X-linked inhibtor of apoptosis protein,XIAP)表达的影响,应用磷脂酰肌醇3激酶(phosphatidyl inositol 3 kinase,P13K)抑制剂LY294002进一步探讨rHuEPO作用的可能机制.方法 采用PTZ制作大鼠SE模型,将点燃后的大鼠随机分为PTZ组(PTZ+NS)、rHuEPO组(PTZ+rHuEPO)、LY294002组(PTZ+LY294002+rHuEPO)、LY294002溶剂二甲基亚砜(DMSO)对照组(PTZ+DMSO+rHuEPO),正常对照组腹腔注射生理盐水(n=35).观察大鼠行为学和脑电图的改变;TUNEL法检测海马神经细胞的凋亡情况;免疫组织化学法观察p-Akt、XIAP的表达;RT-PCR法检测各组大鼠海马XIAPmRNA的表达;Western blot法检测各组大鼠海马Akt、P-Akt蛋白的表达.结果 正常对照组仅见少量凋亡细胞,p-Akt和XIAP阳性细胞、p-Akt蛋白、XIAP mRNA均有少量表达;PTZ组与rHuEPO组、LY294002组、LY294002溶剂DMSO对照组比较,p-Akt和XIAP阳性细胞数、P-Akt蛋白表达及XIAP mRNA表达均减少(P<0.05),凋亡细胞数增加(P<0.05);rHuEPO组、LY294002溶剂DMSO对照组与LY294002组比较,p-Akt和XIAP阳性细胞数、p-Akt蛋白表达及XIAP mRNA表达均增加(P<0.05),凋亡细胞数减少(P<0.05).结论 rHuEPO在SE模型中活化了PI3K/Akt,提高p-Akt蛋白的表达,进而对线粒体凋亡途径的相关调控因子XIAP的表达进行了调控,从而介导线粒体凋亡途经,发挥抗凋亡、促存活的神经保护作用.     

Trichostatin A sensitizes human ovarian cancer cells to TRAIL-induced apoptosis by down-regulation of c-FLIPL via inhibition of EGFR pathway

TRAIL-resistant cancer cells can be sensitized to TRAIL by combination therapy. In this study, we investigated the effect of trichostatin A (TSA), a histone deacetylase inhibitor, to overcome the TRAIL resistance in human ovarian cancer cells. Co-treatment of human ovarian cancer cells with TSA and TRAIL synergistically inhibited cell proliferation and induced apoptosis. The combined treatment of ovarian cancer SKOV3 cells with TSA and TRAIL significantly activated caspase-8 and truncated Bid, resulting in the cytosolic accumulation of cytochrome c as well as the activation of caspase-9 and -3. Moreover, we found that down-regulation of c-FLIP_L might contribute to TSA-mediated sensitization to TRAIL-induced apoptosis in SKOV3 cells, and this result was supported by showing that down- or up-regulation of c-FLIP_L with transfection of siRNA or plasmid sensitized or made SKOV3 cells resistant to TRAIL-induced apoptosis, respectively. TSA or co-treatment with TSA alone and TRAIL also resulted in down-regulation of EGFR1/2 and dephosphorylation of its downstream targets, AKT and ERK. Treatment of SKOV3 cells with PKI-166 (EGFR1/2 inhibitor), LY294002 (AKT inhibitor), and PD98059 (ERK inhibitor) decreased c-FLIP_L expression and co-treatment with TRAIL further reduced the level of c-FLIP_L, respectively, as did TSA. Collectively, our data suggest that TSA-mediated sensitization of ovarian cancer cells to TRAIL is closely correlated with down-regulation of c-FLIP_L via inhibition of EGFR pathway, involving caspase-dependent mitochondrial apoptosis, and combination of TSA and TRAIL may be an effective strategy for treating TRAIL-resistant human ovarian cancer cells.     

Smac和XIAP基因在肾细胞癌中的表达及意义

目的 探讨Smac和XIAP基因在肾细胞癌组织中的表达及其临床意义.方法 采用免疫组织化学法检测95例肾癌标本不同分型、分级、分期及10例正常肾组织中Smac和XIAP的表达,分析其表达与肾癌分级、分期的关系及二者表达相关性.结果 Smac在各型肾细胞癌中随分期、分级的增加表达减低,XIAP在各型肾细胞癌中随分期、分级的增加表达增高,二者的表达呈负相关.结论 Smac和XIAP表达与肾细胞癌分期、分级关系密切,共同参与了肾癌的发生发展. Abstract： Objective To study the expression and significance of Smac and XIAP in renal cell carcinoma(RCC). Methods The expressions of Smac and XIAP were detected using SP immunohistochemical technique in 95 cases of RCC and 10 cases of normal renal tissue. Results The postitive expressions of Smac were statistically significant differences in different clinical statges. The postitive expressions of XIAP were higher in RCC related to the lower histological grade. Smac protein and XIAP protein expression had negative correlation. Conclusions It indicate that Smac and XIAP may play an important role in RCC. The expression of Smac and XIAP are statistically significant associated with tumor grade and clinical stage.     

XIAP siRNA对TRAIL诱导结肠癌细胞SW620凋亡能力的影响

 目的
研究XIAP siRNA对肿瘤坏死因子相关的凋亡诱导配体(Tumor Necrosis Factor-related Apoptosis-Inducing Ligand,TRAIL)诱导结
肠癌细胞SW620凋亡的影响。方法SW620细胞分为转染XIAP siRNA组、空转染对照组或者siRNA阴性对照组,然后给予TRAIL处理。XIAP
表达水平的变化、细胞增殖抑制、Caspase-3活性分别由Western blot、MTT法和Caspase-3荧光测定来检测。切割PARP的表达水平由
Western blot来测定。结果XIAP siRNA转染以后显著下调XIAP蛋白表达水平。和空转染对照组、siRNA阴性对照组相比,XIAP siRNA
显著增强10、100、1 000 ng/ml等浓度的TRAIL作用以后SW620细胞的增殖抑制、Caspase-3活性和激活PARP。结论 XIAP siRNA能显
著增强TRAIL诱导结肠癌细胞SW620的凋亡。     

Rocaglamide and a XIAP inhibitor cooperatively sensitize TRAIL-mediated apoptosis in Hodgkin's lymphomas

Although most of the patients with Hodgkin's lymphoma (HL) can be cured by the current regimen of high-dose multiagent chemotherapy, the treatment causes high risks of later toxicities including secondary malignancies. Therefore, new rational strategies are needed for HL treatment. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent due to its tumor selectivity and its lack of toxicity for normal cells. Unfortunately, many cancers remain resistant to TRAIL including HL. HL is characterized by enhanced expression of cellular caspase-8 (FLICE)-inhibitory protein (c-FLIP) and X-linked inhibitor of apoptosis (XIAP), which block receptor-mediated apoptosis by inhibiting caspase-8 and caspase-3, respectively. We have recently discovered the herbal compound Rocaglamide, which breaks TRAIL-resistance in acute T cell leukemia through inhibition of c-FLIP expression. We have also shown that small molecule XIAP inhibitors can sensitize TRAIL-mediated apoptosis in several resistant tumors. However, whether targeting XIAP or c-FLIP is also a suitable strategy to prime HL cells for TRAIL-induced apoptosis has not yet been investigated. In our study, we show that Rocaglamide suppresses c-FLIP expression in HL cells in a dose- and time-dependent manner. However, downregulation of c-FLIP alone was not sufficient to sensitize TRAIL-induced apoptosis in HL cells. Similarly, treatment of HL cells with a small molecule XIAP inhibitor resulted in a moderate induction of apoptosis. However, inhibition of XIAP alone was also not sufficient to enhance TRAIL-induced cell death. Synergistic increase in TRAIL-mediated killing of HL cells was only obtained by combination of Rocaglamide and XIAP inhibitors. Our study demonstrates that targeting both c-FLIP and XIAP are necessary for an efficient treatment of HL.     

抗凋亡基因XIAP在慢性淋巴细胞白血病中的异常表达及其临床意义

目的: 慢性淋巴细胞白血病(chronic lymphoctyic leukemia,CLL)存在多种因素导致的细胞凋亡受抑,本文主要探讨抗凋亡基因XIAP在慢性淋巴细胞白血病中的表达,并深入分析其与IgVH突变?CD38等临床重要预后指标的相关性?方法:本研究采用SyberGreen法实时定量PCR(qPCR)技术检测37例CLL患者肿瘤细胞中抗凋亡基因XIAP的mRNA表达水平,同时利用28例年龄匹配的正常人作为阴性对照,分析其临床意义?结果:经过qPCR检测正常对照组XIAP表达中位数为0.04040(95% CI:0.01610~0.06471),CLL组XIAP表达为0.07502(0.04383~0.1062),两者具有显著统计学差异(P=0.0025);亚组分析发现耐药组XIAP表达为0.08295(0.02575~0.1402),药物敏感组为0.07020(0.03042~0.1100),耐药组XIAP表达高于药物敏感组,但是未达到统计学差异(P=0.3978)?进一步分析显示XIAP mRNA表达水平和IgVH突变状态和CD38表达相关:IgVH无突变和CD38高表达的CLL患者XIAP表达显著升高(P值分别为0.0265和0.0388)?结论:利用qPCR技术发现抗凋亡基因XIAP在CLL患者中存在异常高表达,耐药组有高于药物敏感组的趋势,并且与CLL临床预后因素IgVH突变状态和CD38表达密切相关,提示其可能成为预测CLL预后的新指标?