siRNA-survivin与GRIM-19共表达质粒的构建及其对前列腺癌DU145细胞生长的抑制作用

目的: 构建siRNA-survivin与GRIM-19共表达质粒pGRIM-19-si-survivin并鉴定,观察pGRIM-19-si-survivin质粒对人前列腺癌DU145细胞survivin和GRIM-19 mRNA表达水平及细胞增殖能力的影响。方法:根据前期工作,利用基因重组技术构建siRNA-survivin与GRIM-19共表达质粒,将成功构建的pGRIM-19-si-survivin质粒转染人前列腺癌DU145细胞株,应用半定量RT-PCR方法检测细胞内survivin和GRIM-19 mRNA表达水平,应用MTT法观察其对细胞增殖能力的影响。结果:(1)应用酶切及质粒测序法鉴定,证明成功构建siRNA-survivin与GRIM-19共表达质粒。(2)与mock组比较,psi-survivin、pGRIM-19及pGRIM-19-si-survivin组survivin mRNA表达水平分别为0.55±0.05、0.62±0.08和0.35±0.05,差异显著(P<0.01);与psi-survivin和pGRIM-19组比较,pGRIM-19-si-survivin的抑制survivin表达作用明显增强(P<0.05);psi-survivin、pGRIM-19和pGRIM-19-si-survivin组GRIM-19表达增强,分别为mock组的1.93±0.14、2.57±0.20 和4.12±0.21(P<0.01),与pGRIM-19组比较,pGRIM-19-si-survivin增强GRIM-19表达作用更明显(P<0.05)。(3)转染48 h后,与mock组比较,psi-survivin、pGRIM-19和pGRIM-19-si-survivin 3组细胞增殖率分别为58.0%±7.2%、62.1%±6.1%和50.2%±4.8%,差异显著(P<0.05);转染72 h后,与mock组比较,3组细胞增殖率分别为43.4%±4.3%、51.3%±6.7%和26.8%±7.1%,差异明显(P<0.05,P<0.01);与psi-survivin和pGRIM-19组比较,pGRIM-19-si-survivin抑制作用明显增强(P<0.05)。结论:成功构建siRNA-survivin和GRIM-19共表达质粒pGRIM-19-si-survivin;该质粒抑制survivin表达并增强GRIM-19表达,对前列腺癌DU145细胞具有协同增殖抑制效应。     

携带pGRIM-19-si-survivin共表达质粒的减毒沙门菌对前列腺癌移植瘤的体内抑制作用

目的:探讨人减毒沙门菌携带pGRIM-19-si-survivin共表达质粒对人前列腺癌皮下移植瘤生长的抑制作用及相关机制。方法:复制裸鼠前列腺癌皮下移植瘤模型,通过腹腔注射携带共表达质粒的减毒沙门菌进行治疗,实时监测肿瘤体积;运用免疫组织化学染色法、RT-PCR法和TUNEL法检测携带pGRIM-19-si-survivin共表达质粒的减毒沙门菌对肿瘤抑制作用的相关机制。结果:与psi-survivin或pGRIM-19单基因治疗对照组比较,携带pGRIM-19-si-survivin共表达质粒减毒沙门菌组肿瘤体积明显缩小,其缩小率分别约为2.36和3.02倍;肿瘤组织内survivin mRNA及蛋白表达明显下降(P0.05),GRIM-19 mRNA及蛋白表达明显增高(P0.05),凋亡相关蛋白Bcl-xL、Stat3、cyclin D1和c-Myc的mRNA表达明显降低(P0.05),血管内皮生长因子(VEGF)mRNA及Ki67蛋白表达降低而caspase-3 mRNA表达明显上调(P0.05),同时细胞凋亡明显。结论:携带pGRIM-19-si-survivin共表达质粒的减毒沙门菌明显抑制裸鼠前列腺癌皮下移植瘤的生长,其发生机制与促进细胞凋亡及抑制肿瘤细胞增殖有关。     

左炔诺孕酮下调survivin基因促进人子宫肌瘤细胞凋亡

目的探讨左炔诺孕酮(LNG)诱导人子宫肌瘤细胞(UtLMC)凋亡过程中survivin的表达变化。方法原代培养人子宫肌瘤细胞传代后,加入不同浓度LNG,以AO/EB双染法区分早、晚期凋亡细胞和坏死细胞;RT-PCR检测抑凋亡基因survivin mRNA的表达,Western blot测定survivin蛋白的表达。结果 10 mg/L LNG作用UtLMC后早期凋亡细胞多于阴性对照组,随着LNG剂量的增加,晚期凋亡细胞也逐渐增多,核浓聚、偏位,被染成桔红色;在10 mg/L以上LNG诱导的UtLMC凋亡中,survivin mRNA表达显著下降,蛋白表达由对照组的33.82±0.02下降至10.37±0.03(P<0.05)。结论一定浓度的左炔诺孕酮所诱导的人子宫肌瘤细胞凋亡,可能与抑制survivin抗凋亡活性相关。     

Survivin反义寡核苷酸对骨肉瘤细胞凋亡的诱导效应

目的：利用骨肉瘤OS-732细胞，探讨survivin反义寡核苷酸（ASODN）对survivin基因表达的影响及其诱导肿瘤细胞凋亡的效应。 方法: 设计合成特异性靶向survivin的ASODN，以不同浓度和时间对OS-732细胞进行转染，并设空白、正义（SODN）对照组进行比较。采用RT-PCR、Western blotting、免疫细胞化学技术检测各组OS-732细胞中survivin mRNA、蛋白表达状态，吖啶橙/溴化乙锭（AO/EB）染色法、流式细胞仪检测细胞凋亡水平和形态变化，MTT法检测细胞生长抑制情况，激酶活性测定法检测细胞中caspase-3活性程度。 结果: 转染ASODN各组OS-732细胞survivin mRNA和蛋白表达均明显弱于空白对照组、SODN组；细胞凋亡率（AI）显著增加，细胞生长相应受抑，caspase-3活性显著提高；上述效应在ASODN各组存在一定的时间浓度依赖性；而SODN各组与空白对照组间各项指标均无明显差异。 结论: ASODN能够特异性下调OS-732细胞中survivin基因表达，激活凋亡效应蛋白酶caspase-3，在诱导肿瘤细胞凋亡、抑制增殖中发挥重要作用。     

尼古丁抑制穿孔素/颗粒酶B诱导的A549肺癌细胞凋亡

目的: 首次探讨尼古丁对穿孔素/颗粒酶B(perforin/granzyme B)诱导的A549肺癌细胞凋亡的抑制作用及其机制。 方法: 以人肺癌细胞株A549为研究对象,采用四甲基偶氮噻唑蓝(MTT)比色法检测细胞活性,吖啶橙/溴化乙啶(AO/EB)双染荧光显微镜下观察细胞凋亡形态学变化,异硫氰酸荧光素标记的膜联蛋白V/碘化丙啶(Annexin V-FITC/PI)染色后流式细胞术定量检测细胞凋亡率,RT-PCR法检测X连锁凋亡抑制蛋白(XIAP)及存活素(survivin)mRNA的表达量。结果: (1)不同浓度尼古丁单独作用A549细胞时未见明显诱导凋亡现象,可显著抑制细胞生长(P< 0.01),其抑制率与作用时间和剂量呈正相关。(2)0.125 mg/L perforin作用于A549细胞(1×10⁶个)时,PI染核率可达到90%以上。而当加入1 μL granzyme B(1 mg/L)孵育6 h时,perforin/granzyme B组细胞可呈现90%以上的凋亡现象。(3)6 μmol/L尼古丁作用于perforin/granzyme B预处理的细胞6 h时可见明显凋亡抑制现象,当检测其凋亡抑制蛋白XIAP和survivin mRNA的含量时,发现尼古丁组表达量较高,perforin/granzyme B组表达量明显降低,而当尼古丁联合perforin/granzyme B时表达量最高。结论: 尼古丁可明显抑制perforin/granzyme B诱导的A549肺癌细胞凋亡现象,这种作用可能与凋亡抑制蛋白XIAP和survivin的表达上调有关。     

Overexpression of GRIM-19, a mitochondrial respiratory chain complex I protein,suppresses hepatocellular carcinoma growth

GRIM-19 has been demonstrated as an important regulator for the normal tissue development. Recently, more evidences regarded GRIM-19 as the new tumor suppressor. However, the possible mechanisms underlying GRIM-19 suppressing cancer growth are unclear. In the present study, Paired hepatocellular carcinoma (HCC) and adjacent non-tumor liver tissues were obtained from 54 patients who underwent primary surgical HCC tissue resection. GRIM-19 protein expression in HCC tissues was performed by immunohistochemistry. Cells were transfected by lentiviruses plasmid expressing GRIM-19. RT-PCR and Western blot analyses were performed to confirm the expression of GRIM-19 mRNA or protein. Cell proliferation was assessed by MTT and FCM analyses. Mitochondrial membrane potential and apoptosis were respectively determined by using fluorescence microscopy and FCM analyses. AKT1, pAKT1, cyclinD1, CDK4, PCNA, Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3, and cytochrome C were detected by Western blot and immunofluorescence. GRIM-19 protein expression was markedly lower in HCC than in paired adjacent non-tumor liver tissues. GRIM-19 overexpression in HCC cells significantly induced cell cycle arrest and enhanced apoptosis. We also found that AKT1 expression and phosphorylation were regulated by the expression of GRIM-19. Collectively, our study demonstrated that GRIM-19 overexpression suppressed HCC growth and downregulated AKT1 expression, suggesting that GRIM-19 might play a crucial role in hepatocarcinogenesis through negatively regulating the PI3K/AKT signaling pathway.     

尾叶香茶菜二萜类化合物B对小鼠前列腺癌细胞细胞周期的影响及其机制

目的：观察尾叶香茶菜二萜类化合物B(DB)对小鼠前列腺癌细胞株RM-1细胞周期的影响并分析其机制。方法：MTT法观察不同时间、不同剂量的DB对RM-1 细胞增殖率的影响；相差显微镜观察对RM-1细胞形态影响；流式细胞术分析细胞周期变化；RT-PCR和Western blotting分别观察DB作用后RM-1细胞p53、GRIM-19、STAT3 和细胞周期相关因子CHK1、CDK2 mRNA和蛋白水平的表达。结果：（1）MTT结果：DB可抑制RM-1 细胞的增殖，呈时间、剂量依赖性；（2）形态学观察和流式细胞术检测：经DB处理后的RM-1细胞出现生长抑制，细胞主要阻滞在G1期；（3）RT-PCR结果： DB (2 mg/L,4 mg/L,8 mg/L) 分别作用RM-1细胞12 h、24 h、48 h后，p53、CHK1、GRIM-19 mRNA的含量明显高于对照组，CDK2、STAT3 mRNA的含量明显低于对照组（P<0.05）；（4）Western blotting 结果：DB (2 mg/L,4 mg/L,8 mg/L) 分别作用RM-1细胞12 h、24 h、48 h后，P53、GRIM-19 蛋白表达水平高于对照组，而CDK2蛋白表达水平则低于对照组。结论：DB可抑制RM-1细胞增殖，其机制可能是与细胞周期相关基因p53的上调，CDK2表达下降有关，影响了G1期进入S期，同时GRIM-19-STAT3-CDK2途径可能也参与G1期阻滞的过程。     

干扰素/维甲酸联合应用诱导细胞凋亡相关基因-19在小鼠围着床期子宫内膜中的表达及其作用

目的 探讨干扰素/维甲酸联合应用诱导细胞凋亡相关基因-19（GRIM-19）在小鼠围着床期子宫内膜中的表达及其与胚胎着床的关系。方法 收集早期妊娠第1~6天小鼠子宫组织,采用免疫组织化学法检测GRIM-19蛋白在子宫内膜上皮细胞中的表达及定位情况;收集早期妊娠、假孕和雌孕激素处理小鼠子宫组织,采用Western blotting 和Real-time PCR检测GRIM-19蛋白和mRNA水平;TUNEL方法检测早期妊娠第1~6天小鼠子宫组织细胞凋亡情况;采用pEGFP GRIM-19质粒GRIM-19-siRNA 转染技术使RL95-2细胞系过表达或低表达GRIM-19,检测其对RL95-2-BeWo 共培养体外着床模型球状体黏附率的影响,AnnexinV/PI双染色法检测细胞凋亡,线粒膜电位检测试剂盒（JC-1）检测线粒体跨膜电位;采用Western blotting 和Real-time PCR检测转染后信号转导子和转录激活因子3（STAT3）、肿瘤坏死因子（TNF)-α及白细胞介素（IL)-11蛋白和mRNA水平。结果 GRIM-19在早期妊娠第1~6天小鼠子宫腔上皮和腺上皮均有表达,妊娠第4天GRIM-19蛋白和mRNA水平减低,细胞凋亡减少;假孕第1~6天小鼠子宫内膜GRIM-19表达无明显差异;雌激素处理组小鼠子宫内膜GRIM-19表达减弱;过表达GRIM-19后,RL95-2-BeWo 共培养球状体黏附率降低,细胞凋亡增加,线粒体膜电位减低,RL95-2细胞系中STAT3及IL-11蛋白和mRNA水平减低,TNF-α蛋白和mRNA水平升高。 结论 GRIM-19在胚胎着床中发挥重要作用,可能是通过调节细胞凋亡和免疫耐受为胚胎着床提供保障。     

特异性干扰survivin 基因表达对人舌癌Tca8113 细胞体外增殖和侵袭性的影响

目的：探讨应用RNAi技术特异性沉默survivin基因表达对人舌癌细胞Tca8113增殖、凋亡、迁移的影响,阐明survivin在Tca8113细胞生物学行为中的作用。方法：取对数生长期的Tca8113细胞,分为干扰组、阴性对照组及空白对照组。采用RT-PCR和Western blot方法检测Tca8113细胞中survivin基因的mRNA及蛋白的表达水平;MTT法检测细胞的增殖状况;流式细胞术检测各组Tca8113细胞凋亡率;划痕实验检测Tca8113细胞的迁移状况。结果：RT-PCR 显示舌癌Tca8113细胞中survivin的mRNA表达水平明显低于空白对照组 (P<0.05),Western blot 蛋白免疫印迹结果显示,与空白对照组比较,survivin蛋白表达水平减少(P<0.05)。survivin干扰组Tca8113细胞的增殖能力降低（P<0.05）;细胞划痕实验发现转染Tca8113细胞48 h 后干扰组细胞的迁移距离明显短于空白对照组( P<0.05)。结论：特异性沉默siRNA-survivin的表达可有效抑制舌癌Tca8113细胞的体外迁移能力,可能与survivin表达减少有关,有望成为口腔舌癌生物治疗的潜在靶点。     

GRIM-19基因的克隆及其对小鼠前列腺癌细胞株的促凋亡作用

目的构建GRIM-19基因真核表达质粒,探讨GRIM-19基因转染对小鼠前列腺癌rm-1细胞的促凋亡作用。方法采用RT-PCR及重组DNA技术构建pcDNA-GRIM-19真核表达质粒,体外进行基因转染。形态学观察,DNAladder检测转染后rm-1细胞凋亡,RT-PCR方法检测rm-1细胞caspase-3活性。结果扩增出GRIM-19cDNA全长,测序结果与GenBank记载基本一致。转染rm-1细胞48h后证明其能在真核细胞表达,形态学及共聚焦显微镜观察到pcDNA3.1-GRIM-19重组质粒组出现明显的细胞凋亡,并出现典型的DNAladder。转染pcDNA3.1-GRIM-19重组质粒组caspase-3表达强度较对照组及空质粒组明显上调(P<0.05)。结论成功克隆并构建鼠GRIM-19基因真核表达载体pcDNA3.1-GRIM-19,该载体可诱导鼠rm-1细胞凋亡。其凋亡机制与caspase-3酶活性有关。     

Celecoxib通过β-catenin-Tcf/Lef通路诱导SW620细胞凋亡的体外研究

目的 研究Celecoxib抗大肠癌的分子生物学机制.方法 不同浓度的Celecoxib作用于大肠癌细胞SW620,MTT(methyl thiazolyl tetrazolium,MTT)法检测细胞增殖,Western印迹检测β-catenin 和survivin蛋白表达的变化,流式细胞仪检测细胞凋亡的变化.siRNA沉默β-catenin,观察survivin蛋白表达变化情况.结果 Celecoxib浓度在10-mmol/L～10-3mmol/L范围内表现出较强的抑制细胞增殖的效应分别为34.13%、68.93%和71.66%,呈剂量依赖性;Celecoxib可抑制β-catenin和survivin蛋白表达;流式细胞仪检测结果显示60 μmol/L和80μmol/L Celecoxib处理后SW620细胞凋亡比例明显增高,凋亡细胞分别为对照组的4.9倍[(x±s)%=(4.02±0.67)%,P=0.007 837]和8.2倍[(x±s)%=(6.50±0.56)%,P=0.004 296];siRNA沉默β-catenin后发现survivin表达显著降低.结论 Celecoxib通过影响β-catenin,进一步通过β-catenin-Tcf/Lef途径抑制survivin表达来起到诱导SW620细胞凋亡的作用.     

Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96''µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12''h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer.     

铁碳纳米粒介导顺铂对喉癌细胞敏感性的研究*

背景：应用纳米技术对化疗药物进行靶向性改进是增加肿瘤对化疗药物敏感性的有效手段之一。 目的：观察铁碳纳米粒搭载顺铂对喉癌Hep-2细胞的抑制作用及对细胞Caspase 3,Survivin mRNA表达的影响。 方法：分别应用铁碳纳米粒和(或)顺铂的生理盐水分散液干预喉癌Hep-2细胞,以生理盐水干预的细胞作为对照。 结果与结论：MTT检测结果显示,顺铂可明显抑制Hep-2细胞的生长,与铁碳纳米粒联合应用对Hep-2细胞的抑制作用更强;表现为细胞生长不贴壁,增殖缓慢,出现凋亡征象。RT-PCR结果显示,共培养5 d,铁碳纳米粒和顺铂联合及顺铂单独处理的Hep-2细胞Caspase 3 mRNA水平明显增高,而Survivin mRNA水平明显降低,以铁碳纳米粒和顺铂联合作用效果更明显,而单独应用铁碳纳米粒对Hep-2细胞无影响。提示铁碳纳米粒搭载顺铂能够增加喉癌Hep-2细胞对顺铂的敏感性,提高药物化疗效果。 关键词：铁碳纳米粒;顺铂;喉癌;Hep-2细胞;化疗;敏感性;Caspase3;Survivin doi:10.3969/j.issn.1673-8225.2012.12.010     

The IFN-beta and retinoic acid-induced cell death regulator GRIM-19 is upregulated during focal cerebral ischemia.

The induction of GRIM-19 has been shown to be essential for interferon-beta (IFN-beta)-induced and retinoic acid (RA)-induced tumor cell death. We have studied the localization and levels of GRIM-19 in IFN/RA-induced cell death in neural cells and in focal cerebral ischemia. Exposure to IFN/RA caused a approximately 15-fold increase in GRIM-19 protein levels and induced >50% cell death in human neuroblastoma SH-SY5Y cells. In rats subjected to permanent focal cerebral ischemia, increased oxidative stress, as well as increased GRIM mRNA levels (32-fold) and increased GRIM-19 (>50%) protein levels were noted in the ipsilateral (affected) hemisphere compared with the contralateral (unaffected) hemisphere. These results suggest that GRIM-19 may play a role in ischemia-induced neuronal cell death.     

Characterization of monoclonal antibodies against GRIM-19, a novel IFN-beta and retinoic acid-activated regulator of cell death.

A combination of interferon-beta (IFN-beta) and all-trans retinoic acid (IFN/RA) induces tumor cell apoptosis via some unknown mechanisms. Apoptosis is a gene-directed process that limits the proliferation of undesired cells. Several genes are required to regulate cell death in the higher-order animals. Earlier, we employed a gene expression knockout technique to isolate cell death-related genes. A novel gene, the gene associated with retinoid-interferon-induced mortality-19 (GRIM-19), was found to be essential for tumor cell death induced by IFN/RA. Here, we describe the development and characterization of three monoclonal antibodies (mAbs) against GRIM-19. GRIM-19 is present in the nucleus and cytoplasm. Its expression is induced by the IFN/RA combination. We also show that GRIM-19 inhibits the cell-transforming property of viral oncogenic protein viral IFN regulatory factor-1 (vIRF-1) via a physical interaction. mAbs developed in this study should be useful for studying the other physiologic roles of GRIM-19 and serve as a potent tool for studying tumor responses to IFN/RA therapy.     

Naked DNA immunization as an approach to target the generic tumor antigen survivin induces humoral and cellular immune responses in mice

Survivin, a 16.5 kDa tumor associated antigen, is the smallest member of the inhibitor of apoptosis family that is abundantly expressed during development but essentially absent in normal adult tissues. Interestingly, survivin expression is up-regulated in virtually all types of cancers studied, as well as in vascular endothelial cells during tumor associated angiogenesis. Survivin links apoptosis to cell cycle progression and plays a pivotal role in regulation of cell proliferation. These characteristics make survivin a potentially promising generic target for cancer immunotherapy. Hence, a genetic immunization strategy to induce tumor-specific immune responses against human survivin in a pre-clinical animal model was developed. In initial studies, BALB/c mice were immunized by intramuscular injection with DNA coding for human survivin (pcDNA3.1/hSurv). In addition, a construct encoding a secreted version of survivin (pSecTag2B/hSurv) was designed. A plasmid coding for murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was co-injected in both cases as a molecular adjuvant. Expression of survivin following transfection in mouse cells was corroborated. Humoral responses against human survivin were detected in mice sera using two immunization protocols (injections at 2- or 3-week intervals). The humoral response was markedly improved by secretion of survivin and co-expression of GM-CSF. The predominant antibody subclass detected in responsive mice was IgG2a, suggesting that a Th1-CD4+ cellular response had been induced. Furthermore, DNA immunization with survivin encoding vectors generated an effective CD8+ T cell response measured as an increase of cytotoxic Interferon-gamma (IFN-gamma) secreting CD8+ T cells. In conclusion, intramuscular genetic immunization of mice with human survivin encoding plasmids induced a survivin-specific humoral as well as cellular immune response in recipient mice. Secretion of survivin and co-injection of GM-CSF as a genetic adjuvant appear to be more important in generating an humoral than a cellular immune response.     

GRIM-19在小鼠植入前胚胎中的表达及其作用

目的 通过研究干扰素/维甲酸联合应用诱导细胞凋亡相关基因 (GRIM-19)在小鼠植入前胚胎中的表达及其与胚胎质量之间的相关性,探讨GRIM-19在小鼠植入前胚胎发育中的作用。方法 采用实时定量PCR,检测小鼠植入前胚胎（2-细胞期、4-细胞期、8-细胞期、桑葚胚和囊胚）中GRIM-19 mRNA水平 (n =16);将小鼠8 细胞期胚胎按卵裂球大小、形态、透明带、胞质碎片分为优胚组和非优胚组,分别检测两组胚胎GRIM-19 mRNA水平,并分析其相关性 (n =13);制作假孕鼠（23只）,并随机分为A组（12只）和B组（11只）,采用移植器将优质和非优质8-细胞期胚胎移入假孕鼠的子宫内,观察两组雌鼠妊娠率及产仔率。结果 GRIM-19在小鼠植入前各期胚胎中均有表达,其mRNA水平从2-细胞期逐渐增高,至8-细胞期达高峰,随后呈下降趋势。优质8-细胞胚胎的GRIM-19 mRNA水平明显高于非优胚组（P <0.05）。两组8-细胞胚胎移植后,A组雌鼠妊娠率及产仔率显著高于B组雌鼠（P<0.05）。 结论 小鼠植入前胚胎中GRIM-19的表达量随发育时程的改变而变化,且与胚胎质量密切相关。     

预适应对氧化应激中HepG2细胞保护作用的初步探讨

目的：建立HepG2细胞预适应、氧化应激模 型。方法:应用不同浓度H₂O₂作用于HepG2细胞，分别用吖啶橙和溴 化乙锭（AO/EB）双染色法、MTT比色法及PI染色流式细胞术检测细胞活力及凋亡情况。结果:各组细胞经AO/EB双染色之后呈现不同的染色状态：对照组细胞为正常 梭形，呈均匀绿色荧光染色；预适应组可见少量绿色浓染细胞；氧化应激组可见大量绿色或 红色浓染凋亡细胞；预适应后氧化应激组凋亡细胞明显少于氧化应激组。预适应组MTT比色 法测定细胞生存活力比较：对照组>预适应组>预适应后氧化应激组>氧化应激组（P<0.05）。PI染色流式细胞术测细胞凋亡率比较：氧化应激组>预适应后氧化应激组>预适应组> 对照组（P<0.05）。结论:不同浓度H₂O₂作用于HepG2细胞发 生预适应和氧化应激现象，应用小剂量H₂O₂作用于细胞可以保护细胞免受更大浓度H₂ O₂带来的损伤。     

PFP、GrB的共表达杀伤人喉癌Hep-2细胞的体外研究

为探讨穿孔素(PFP)、颗粒酶B(GrB)共表达对人喉癌(Hep-2)细胞生长的抑制及其诱导该细胞的凋亡作用,采用RT-PCR的方法从人的喉癌组织浸润淋巴细胞中扩增全长PFP、GrB的cDNA片段,构建共表达重组体pVAX1-PIG,并将其转染入人的Hep-2细胞株中。收集转染后的Hep-2细胞,采用软琼脂集落形成实验、MTT法、原位末端标记法(TUNEL)、流式细胞仪(FCM)检测分析各组人Hep-2细胞的生长抑制及其凋亡情况。结果显示pVAX1-PIG转染组的集落形成数目比空白对照组与pVAX1转染组明显减少(P<0.05),MTT检测结果显示对照组细胞生长速度比pVAX1-PIG转染组要快。TUNEL染色、FCM法检测均显示pVAX1-PIG转染组的Hep-2细胞大量凋亡且其凋亡率显著高于对照组(P<0.05)。因此,PFP、GrB的共表达能够抑制人Hep-2细胞的生长并且可以诱导该细胞的凋亡。