胃癌组织中环氧化酶-2 mRNA及蛋白的表达

[目的]检测环氧化酶-2(Cyclooxygenase-2,COX-2)mRNA及其蛋白在胃癌组织中的表达并探讨其意义.[方法]对60例胃腺癌及对应的癌旁组织中利用RT-PCR法检测COX-2 mRNA的表达,免疫组织化学SP法检测COX-2蛋白的表达.[结果]CO)X-2 mRNA及其蛋白在胃癌组织中的表达阳性率分别为51.7%及48.3%,两者相比差异无显著,且相关分析显示,两检测法的结果呈正相关.而癌旁组织中均不表达COX-2 mRNA及其蛋白,与胃癌组织中相比差异有显著性(P＜0.05).[结论]COX-2的表达与胃癌的发生有关,COX-2 mRNA或其蛋白的检测两者均可反映出胃癌组织中COX-2的表达情况.     

多发性骨髓瘤U266细胞株DNA甲基转移酶和甲基结合蛋白的表达

目的检测多发性骨髓瘤（MM）细胞DNA甲基转移酶（DNMTs）及甲基结合结构域（MBD）蛋白[包括甲基CpG结合结构域蛋白（MeCP2）和MBD2]的表达状况,探索其在基因甲基化过程的作用。并观察5-氮杂-2＇-脱氧胞苷（5-Aza-CdR）和/或曲古霉素A（TSA）对DNMTs及MeCP2和MBD2的作用。方法利用荧光定量聚合酶链反应（PCR）分析MMU266细胞株DNMTs（DNMT1、DNMT3a、DNMT3b）及MeCP2和MBD2蛋白的mRNA水平,与10名正常人单个核细胞（PBMC）mRNA比较。4μmol/L5-Aza-CdR和/或0.1μmol/LTSA与U266细胞共培养,分析MBD2和MeCP2蛋白mRNA的变化情况。WesternBlot检测这2种药物对U266细胞MBD2蛋白的影响。结果各甲基化调控蛋白mRNA与PBMCmRNA相比,差异有统计学意义（P均〈0.05）。其中DNMTs和MBD2蛋白在U266细胞中表达升高,而MeCP2蛋白表达下降。经5-Aza-CdR和/或TSA作用后,MBD2表达下降,MeCP2升高。5-Aza-CdR在蛋白水平对MBD2的作用亦不显著;TSA可明显降低MBD2的表达,且具有时间依赖性。结论 DNMTs和MBD2、MeCP2蛋白在MMU266细胞中表达异常,可能与U266细胞多个抑癌基因启动子高甲基化相关。5-Aza-CdR和TSA可以逆转U266细胞MBD2和MeCP2的表达。     

NSCLC组织中MMP-9、bcl-2及HSP 90α表达及其临床意义

[目的]探讨非小细胞肺癌(NSCLC)组织中基质金属蛋白酶-9(MMP-9)、B淋巴细胞瘤-2基因(bcl-2)、热休克蛋白90α(HSP90α)的蛋白及其mRNA的表达情况及临床意义.[方法]收集62例NSCLC患者的病理组织标本作为观察组,并取其中30例患者癌旁正常肺组织(距肿瘤边缘>3 cm)作为对照组,测定两组MMP-9、bcl-2、HSP90α蛋白及其mRNA表达水平及与临床病理特征的相关性.[结果]①观察组MMP-9 mRNA、bcl-2 mRNA、HSP90αmRNA蛋白表达水平及阳性率均高于对照组(P<0.05);②不同TNM分期、伴或不伴淋巴结转移NSCLC患者组织MMP-9、HSP90α蛋白阳性率比较差异有显著性(P<0.05),不同组织类型NSCLC患者组织bcl-2蛋白阳性率比较差异有显著性(P<0.05).[结论]NSCLC组织中MMP-9、bcl-2、HSP90α蛋白阳性率均高于癌旁正常组织,MMP-9、bcl-2表达与TNM分期及淋巴结转移有关,bcl-2蛋白表达与NSCLC组织学类型有关.     

RUNX3在人胆管癌组织中的表达及临床意义

[目的]探讨人胆管癌组织中相关转录因子3( RUNX3)mRNA 、RUNX3蛋白的表达及与胆管癌临床病理因素的关系及临床意义.[方法]用反转录聚合酶链反应(RT-PCR)分别检测20例手术切除的胆管癌组织及胆管炎组织中RUNX3mRNA的表达;用免疫组织化学方法(SP法)检测45例胆管癌及10例正常胆管中RUNX3蛋白的表达.[结果]①在癌组织中RUNX3mRNA表达明显低于胆管炎组织,且两者相比差异有显著性(P<0.01).②胆管癌细胞中RUNX3蛋白表达明显低于正常胆管细胞且两者相比差异有显著性(P<0.01).③不同性别、年龄的胆管癌组织中RUNX3蛋白的表达相比较无显著性差异(P>0.05).④癌细胞分化程度越低、有淋巴转移及临床病理分期(UICC分期)越晚的胆管癌组织中,RUNX3蛋白表达越低(P<0.01).[结论]①在人胆管癌组织中RUNX3的表达下调.②在人胆管癌组织中RUNX3蛋白的表达与胆管癌患者的性别、年龄以及肿瘤部位无关,与癌细胞分化程度、淋巴转移及临床病理分期密切相关.     

HSF1在热休克反应中对KLF4基因表达的影响

[目的]观察热休克因子1(HSF1)在热休克反应中对Kruppel 样因子4(KLF4)基因表达的影响;采用生物信息学方法初步探讨KLF4在热休克反应中调控的下游基因.[方法]采用HSF1基因敲除小鼠热休克模型,抽提HSF1基因敲除小鼠(HSF1^-/-)和野生型小鼠(HSF1^ / )心肌及肺组织的总RNA进行RT-PCR和Northern blot实验,观察KLF4mRNA表达的情况.用热休克处理和HSF1过表达的小鼠RAW264.7巨噬细胞,抽提总RNA进行RT-PCR实验,观察KLF4mRNA表达的情况;用TESS分析启动子含有KLF4结合位点的下游基因.[结果]热休克处理后,HSF1^ / 小鼠组织中KLF4mRNA的水平明显增加,HSF1^-/-小鼠组织中KLF4 mRNA水平的增加明显低于HSF1^ / 小鼠.小鼠RAW264.7巨噬细胞受热刺激后,KLF4mRNA的水平明显增加;在HSF1过表达细胞中KLF4的表达也明显增高.经TESS软件分析发现6个启动子区含有KLF4结合位点的下游基因.[结论]HSF1诱导KLF4基因在热休克反应中呈现高表达.     

ACTL6A在骨肉瘤组织中的表达及其意义

[目的]探讨核内的肌动蛋白样-6a(Actin-like 6A,ACTL6A)在骨肉瘤组织中的表达及其意义.[方法]采用实时定量PCR、蛋白质免疫印迹(Western Blot,WB)检测ACTL6A mRNA及蛋白在骨肉瘤组织、邻近肿瘤的非瘤骨组织、肺转移结节中的表达水平并比较.[结果]骨肉瘤组织中的ACTL6A mRNA及蛋白表达水平均高于相应的邻近肿瘤非瘤骨组织中的表达水平(P<0.05);Ⅲ期骨肉瘤患者ACTL6A mR-NA及蛋白表达水平显著高于Ⅰ/Ⅱ期患者(P<0.05);同一患者其肺转移结节、原发骨肉瘤组织、邻近肿瘤的非瘤骨组织中ACTL6A mRNA及蛋白表达水平依次递减(P<0.05).[结论]ACTL6A在骨肉瘤组织呈高表达,且其高表达与肺转移相关,提示ACTL6A表达水平可作为预测骨肉瘤预后的指标.     

MeCP2和TNC在子宫肌瘤组织中的表达水平及其临床意义

【目的】探讨甲基化CpG结合蛋白2(MeCP2)和肌腱蛋白C(TNC)在子宫肌瘤组织中的表达水平及其临床意义。【方法】选取2019年1月至2021年9月陕西省康复医院收治的109例子宫肌瘤患者为观察组,另选取68例同期因子宫脱垂行子宫切除术患者为对照组。收集两组患者基本资料,采取免疫组化法检测观察组子宫肌瘤组织、子宫肌瘤旁组织及对照组子宫平滑肌组织中MeCP2和TNC蛋白表达情况。分析子宫肌瘤患者子宫肌瘤组织及瘤旁组织中MeCP2和TNC蛋白表达情况,分析子宫肌瘤发生的影响因素,分析观察组MeCP2和TNC水平与子宫肌瘤直径关系。【结果】子宫肌瘤组织中MeCP2、TNC阳性表达率均高于子宫肌瘤旁组织(P<0.05)。Logistic多因素回归分析结果显示月经紊乱(OR:3.892,95%CI:1.576~9.611)、流产次数(OR:4.293,95%CI:1.739~10.601)、MeCP2阳性(OR:4.495,95%CI:2.218~9.110)、TNC阳性表达(OR:4.341,95%CI:2.142~8.796)是子宫肌瘤发生的影响因素(P<0.05)。子宫肌瘤≥5 cm患者MeCP2、TNC阳性表达率均高于子宫肌瘤<5 cm患者(P<0.05)。【结论】子宫肌瘤发生与子宫平滑肌组织中MeCP2、TNC表达有关,且其表达水平可能与子宫肌瘤直径相关。     

ROCK1在结直肠癌组织中表达及意义研究

[目的]研究Rho相关卷曲螺旋形成蛋白激酶1(ROCK-1)在结直肠癌组织中的表达及其意义.[方法]测定200例结直肠癌及其癌旁组织ROCK1表达及其mRNA相对表达量,比较不同组织ROCK1阳性表达率及其mRNA相对表达量,并分析结直肠癌组织ROCK1表达与临床病理特征的关系.[结果]结直肠癌组织ROCK1阳性表达率、ROCK1 mRNA表达分别为70.00%、(0.935±0.253)显著高于癌旁组织的8.00%、(0.234±0.128),且差异有显著性(P<0.05);不同性别、年龄、肿瘤直径、分化程度结直肠癌患者ROCK1阳性率比较差异均无显著性(P>0.05);不同TNM分期、淋巴结转移、远处转移患者ROCK1阳性表达率比较差异有显著性(P<0.05).[结论]结直肠癌组织中ROCK1高表达,且ROCK1表达与结直肠癌TNM分期、淋巴结转移及远处转移有关.     

高迁移率族蛋白B1在小鼠巨细胞病毒感染小鼠内耳中的表达

目的:探讨高迁移率族蛋白B1(high mobility group box chromosomal protein 1,HMGB-1)在小鼠巨细胞病毒(murine cytomegalovirus,MCMV)感染导致听力损失发病机制中的作用。方法:采用新生小鼠颅内注射病毒法建立MCMV感染动物模型,测定2周龄小鼠的听性脑干反应(auditory brainstem response,ABR),采用RT-PCR检测耳蜗组织HMGB-1mRNA表达,均与注射生理盐水的对照组比较。结果:ABR,病毒组(28.42±3.03)dB,对照组(15.66±0.38)dB(t=3.042,P<0.01)。HMGB-1mRNA,病毒组2.372±0.193,对照组0.754±0.322(t=7.537,P<0.01)。结论:HMGB-1可能通过维持并加重内耳的迟发性炎症,成为巨细胞病毒感染导致听力损失发生发展的病理基础。     

小鼠肝组织NTCP表达与肝纤维化程度相关性的研究

目的观察不同肝纤维化程度小鼠肝组织中Na~+-牛磺胆酸共转运多肽(NTCP)表达水平的变化,探讨NTCP表达与肝纤维化严重程度的相关性。方法经口灌胃方法给予10%CCl4油剂建立小鼠肝纤维化模型;在CCl4给药不同时间点取肝组织进行Masson及天狼猩红染色,判断肝纤维化程度;实时荧光定量PCR方法检测不同程度肝纤维化小鼠肝组织中NTCP mRNA水平;Western blot方法检测不同程度肝纤维化小鼠肝组织中NTCP蛋白表达水平。结果与正常对照小鼠相比,肝纤维化程度评分为F1~F2的小鼠肝组织中NTCP mRNA及蛋白表达的水平均有显著增加;而与肝纤维化程度评分为F1~F2的小鼠相比,肝纤维化程度评分为F3~F4的小鼠肝组织中NTCP mRNA及蛋白的表达水平则显著下降。结论 NTCP的表达在肝纤维化发生早期升高,在肝纤维化晚期降低,其表达水平的动态变化有可能提示肝纤维化的发展进程。     

雌二醇对NZB/wF1狼疮鼠脾脏树突状细胞雌激素受体表达的影响

目的 通过检测系统性红斑狼疮（SLE）模型鼠脾脏树突状细胞（DC）中雌激素受体（ER）的表达及对体外雌激素的反应性是否异常,探索雌激素调控DC参与SEE发病的机制.方法 红细胞裂解法收集SLE模型鼠—NZB/wF1雌鼠及BALB/c雌鼠的脾脏单个核细胞,抗小鼠CD11c单抗标记磁珠纯化DC,以无酚红RPMI-1640及葡聚糖-活性炭处理的新生牛血清体外培养DC,加入不同浓度10-8 mol/L,10-7 mol/L,10-6 mol/L的雌二醇（E2）体外处理24h,RT-PCR检测DC胞内ER的mRNA水平.结果 两种小鼠脾DC均表达ERα,但不表达ERβ;NZB/wF1雌鼠脾DC的ERα基础表达显著高于同周龄的BALB/c雌鼠（0 mol/L组P＜0.05）;低浓度10-8 mol/L E2显著增加两种鼠脾DC的ERα表达（NZB/wF1鼠P＜0.05,BALB/c鼠P＜0.05）,中浓度10-7 mol/L（NZB/wF1鼠P＜0.05,BALB/c鼠P＜0.05）和高浓度10-6 mol/L（NZB/wF1鼠P＜0.05,BALB/c鼠P＜0.05）E2显著降低两种鼠脾DC的ERα表达.结论 ER表达在狼疮鼠中存在明显异常,不同剂量雌激素对其受体调控作用不同,提示雌激素可能通过调控DC的ER参与SEE发病.     

Overexpression of X-linked inhibitor of apoptosis protein protects against noise-induced hearing loss in mice

Apoptosis is responsible for cochlear cell death induced by noise. Here, we show that transgenic (TG) mice that overexpress X-linked inhibitor of apoptosis protein (XIAP) under control of the ubiquitin promoter display reduced hearing loss and cochlear damage induced by acoustic overstimulation (125?dB sound pressure level, 6?h) compared with wild-type (WT) littermates. Hearing status was evaluated using the auditory brainstem response (ABR), whereas cochlear damage was assessed by counts of surviving hair cells (HCs) and spiral ganglion neurons (SGNs) as well as their fibers to HCs. Significantly smaller threshold shifts were found for TG mice than WT littermates. Correspondingly, the TG mice also showed a reduced loss of HCs, SGNs and their fibers to HCs. HC loss was limited to the basal end of the cochlea that detects high frequency sound. In contrast, the ABRs demonstrated a loss of hearing sensitivity across the entire frequency range tested (2-32?kHz) indicating that the hearing loss could not be fully attributed to HC loss alone. The TG mice displayed superior hearing sensitivity over this whole range, suggesting that XIAP overexpression reduces noise-induced hearing loss not only by protecting HCs but also other components of the cochlea.     

Reciprocal expression of interferon gamma or interleukin 4 during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets

We purified poly(A)+ mRNA from the spleen and lymph nodes at designated times after infection with Leishmania major in genetically susceptible BALB/c and resistant C57BL/6 mice. The steady-state levels of IL-2, IFN-gamma, IL-4, and IL-1 beta mRNA were determined using Northern hybridizations. IL-2 mRNA levels in the infected organs of BALB/c and C57BL/6 mice were comparable after infection, but IFN-gamma and IL-4 mRNA levels were reciprocally expressed. Levels of IFN-gamma mRNA in C57BL/6 draining nodes and spleen were significantly greater than in BALB/c mice except at 4 and 6 wk of infection, when splenic IFN-gamma mRNA levels were transiently comparable. In contrast, IL-4 mRNA was apparent only in BALB/c and not in C57BL/6 nodes and spleen. Tissue levels of IL-1 beta mRNA were 10-20-fold greater in BALB/c mice. BALB/c mice were pretreated with GK1.5 mAb, a manipulation that promotes healing of subsequent infection by transiently depleting L3T4+ cells. At 8 wk of infection, by which time lymphoid organs were repopulated with L3T4+ cells, GK1.5-pretreated BALB/c mice produced IFN-gamma, but not IL-4 message. Serum levels of IgE were markedly elevated in infected BALB/c, but not in infected C57BL/6 or GK1.5-pretreated BALB/c mice, consistent with in vivo biologic activity of IL-4 in nonhealing mice. Treatment of infected BALB/c mice with neutralizing anti-IL-4 antibody abolished the elevation of serum IgE and significantly attenuated the progression of disease as assessed by size and ulceration of the lesion, and by reduction in the number of tissue parasites. Both protective and deleterious responses to Leishmania infection have previously been shown to be L3T4+ cell dependent. Our findings are consistent with the differential expansion of protective, IFN-gamma-producing Th1 cells in healing mice, and the expansion of deleterious, IL-4-producing Th2 cells in nonhealing mice. The inverse relationship of IFN-gamma and IL-4 gene expression during leishmaniasis may underlie the divergence of cellular and humoral immunity that occurs during chronic infection with Leishmania and possibly other intracellular parasites.     

胰岛素急性刺激对 PI3K／Akt 和 ERK 通路的分子调节作用

【目的】研究胰岛素（insulin）急性刺激对PI3K/Akt和ERK信号的调节作用及其意义。【方法】HepG2细胞、6只8周龄BALB/C雄性小鼠分别都给予急性胰岛素刺激和磷酸盐缓冲液（PBS）刺激,细胞提取总蛋白、小鼠提取肝组织总蛋白用 Western blotting 法分析 Akt磷酸化水平和 ERK 磷酸化水平,并用免疫沉淀法检测TERT磷酸化水平。【结果】胰岛素急性刺激组 HepG2细胞和肝组织p-Akt、p-ERK水平均较PBS组上调,抑制PI3K/Akt信号通路后 TERT 活性下降。【结论】胰岛素急性刺激可以激活 PI3K/Akt和 ERK信号通路,抑制PI3K/Akt通路可减弱端粒酶活性,这可能为临床肝癌的诊断与治疗提供依据。     

Noninvasive in vivo delivery of transgene via adeno-associated virus into supporting cells of the neonatal mouse cochlea

There are a number of genetic diseases that affect the cochlea early in life, which require normal gene transfer in the early developmental stage to prevent deafness. The delivery of adenovirus (AdV) and adeno-associated virus (AAV) was investigated to elucidate the efficiency and cellular specificity of transgene expression in the neonatal mouse cochlea. The extent of AdV transfection is comparable to that obtained with adult mice. AAV-directed gene transfer after injection into the scala media through a cochleostomy showed transgene expression in the supporting cells, inner hair cells (IHCs), and lateral wall with resulting hearing loss. On the other hand, gene expression was observed in Deiters cells, IHCs, and lateral wall without hearing loss after the application of AAV into the scala tympani through the round window. These findings indicate that injection of AAV into the scala tympani of the neonatal mouse cochlea therefore has the potential to efficiently and noninvasively introduce transgenes to the cochlear supporting cells, and this modality is thus considered to be a promising strategy to prevent hereditary prelingual deafness.     

碱性成纤维细胞生长因子对噪声暴露后豚鼠听力损伤的保护作用

目的观察碱性成纤维细胞生长因子(bFGF)噪声暴露后听力损伤的保护作用及对豚鼠耳蜗热休克蛋白表达的影响。方法制备噪声暴露后豚鼠耳蜗听力损伤模型;应用免疫组化技术ABC法结合听性脑干反应(ABR)阈值检测及研究bFGF对噪声暴露后豚鼠耳蜗热休克蛋白表达的影响。结果bFGF对噪声暴露后豚鼠的ABR阈值有明显的抑制作用。结论bFGF对噪声暴露后豚鼠耳蜗听力损伤具有保护作用,减弱耳蜗热休克蛋白的表达。     

姜黄素对人肝癌细胞株 He pG2巨噬细胞迁移抑制因子表达的影响

[目的]检测人肝癌细胞株HepG2中巨噬细胞迁移抑制因子（MIF）的表达及释放,探讨姜黄素对HepG2中MIF表达的影响。[方法]反转录聚合酶链反应（RT-PCR）及免疫印迹法（Western Blot）检测对照组及不同浓度姜黄素干预组（10,20,30,40,50μmol/L）HepG2中MIF mRNA及蛋白表达水平;ELISA检测对照组及不同浓度姜黄素干预组（10,20,30,40,50μmol/L ）培基中 M IF蛋白水平。[结果]HepG2高表达MIF mRNA及MIF蛋白,各浓度姜黄素干预均可抑制HepG2中MIF mRNA及MIF蛋白的表达,且呈浓度依赖效应（ P ＜0．05）;HepG2可自分泌MIF蛋白,各浓度姜黄素干预均可抑制 HepG2中MIF 蛋白的释放,且呈浓度依赖效应（ P ＜0．05）。[结论]人肝癌细胞株HepG2可表达及释放MIF ;姜黄素在转录和翻译水平能有效抑制M IF的表达及释放,这可能是其发挥抗肿瘤效应的分子机制之一。     

Experimental autoimmune hearing loss is exacerbated in IL-10-deficient mice and reversed by IL-10 gene transfer

Interleukin-10 (IL-10) has an important role in the homeostatic regulation of autoreactive T-cell repertoire. We hypothesized that endogenous IL-10 would regulate the severity of β-tubulin-induced experimental autoimmune hearing loss (EAHL) and that exogenous IL-10 would abrogate it. BALB/c wild-type (WT) and homozygous IL-10-deficient mice (IL-10(-/-)) underwent β-tubulin immunization to develop EAHL; some IL-10 mice with EAHL were administered IL-10 DNA at the peak of EAHL. Auditory brainstem responses were examined over time. EAHL developed progressively in both WT and IL-10(-/-) mice. However, the severity of hearing loss in the IL-10(-/-) mice was significantly greater than that in WT animals. Moreover, disease severity was associated with a significantly enhanced interferon-γ level and loss of hair cells in IL-10(-/-) mice. IL-10 administered to EAHL IL-10(-/-) mice promoted IL-10 expression. Consequently, hearing significantly improved by protecting hair cells in established EAHL. Importantly, IL-10 treatment suppressed proliferation of antigen-specific T-helper type 1 (Th1) cells, and the suppression can be attributed to inducing IL-10-secreting regulatory T cells that suppressed autoreactive T cells. We demonstrated that the lack of IL-10 exacerbated hearing loss, and the exogenous administration of IL-10 improved hearing. Mechanistically, our results indicate that IL-10 is capable of controlling autoimmune reaction severity by suppressing Th1-type proinflammatory responses and inducing IL-10-secreting regulatory T cells.     

乙肝核酸疫苗接种小鼠后特异性抗原表达及CTL应答的动态观察

目的:观察乙肝病毒(hepatitis B virus,HBV)核酸疫苗pCMV-S2S免疫小鼠后,接种局部HBV preS2S抗原的表达及特异性细胞毒性T淋巴细胞(CTL)应答的动态变化。方法:将pCMV-S2S接种到BALB/c小鼠胫前肌,分别以免疫组化法检测接种局部肌肉HBV preS2S抗原表达的动态变化,乳酸脱氢酶(LDH)释放法检测特异性CTL活性的动态变化。结果:小鼠接种pCMV-S2S后,局部肌肉细胞3d后已有少量目的抗原HBV preS2S蛋白表达,4周时表达最强,至6月时仍可检出有preS2S蛋白表达,但表达强度明显减弱。小鼠接种pCMV-S2S3天后,特异性CTL活性平均值最高为21·67%,12周时CTL活性平均值最高为35·14%,6月时仍可检出较弱的CTL活性。结论:HBV核酸疫苗pCMV-S2S能在小鼠体内持续表达特异性抗原及诱生特异性CTL应答。