乌司他丁对大鼠脑缺血再灌注损伤后的保护作用及其机制研究

目的:探究乌司他丁在脑缺血再灌注损伤中的脑保护作用机制。方法:原代分离培养雄性SD大鼠脑皮质细胞,部分细胞经siRNA沉默HSP70基因。细胞先以无糖培养基在低氧条件下培养,12 h后复糖复氧模拟体外缺血再灌注损伤,并实施乌司他丁预处理干预,流式细胞术检测各组细胞的凋亡率,western-blotting检测Bcl-2,Bax,HSP70,JNK和p-JNK蛋白的表达。结果:与对照组比较,模型组脑组织细胞凋亡率明显增多(P0.05)、Bcl-2和Bax的表达量均有上调,Bcl-2/Bax的比值显著降低(P0.01)、HSP70的表达无显著变化;与模型组比较,乌司他丁处理组脑组织细胞凋亡率明显降低(P0.05)、Bax的表达量显著下调(P0.05),Bcl-2/Bax的比值显著上调(P0.05),HSP70的表达显著上调(P0.05),JNK的表达无显著变化、p-JNK则显著下调(P0.05)。HSP70沉默后乌司他丁的脑保护作用消失,对以上蛋白的表达无显著影响。结论:乌司他丁可能是通过上调HSP70表达进而抑制JNK信号转导通路对缺血再灌注引起的脑损伤起保护作用。     

HSP70 和P53 蛋白在甲状腺乳头状癌中的表达 及其与临床病理特征的关系

目的:探讨热休克蛋白70(HSP70)和P53蛋白在甲状腺乳头状癌组织中的表达及其与临床病理特征的关系。方法:应用免疫组织化学Envision法检测35例甲状腺乳头状癌组织、15例甲状腺良性病变组织及15例正常甲状腺组织中HSP70和P53蛋白的表达,分析HSP70和P53蛋白的表达与甲状腺乳头状癌临床病理学特征的关系。结果:甲状腺乳头状癌组织中HSP7O和P53阳性表达率明显高于甲状腺良性病变组织及正常甲状腺组织(P<0.01)。HSP7O在甲状腺乳头状癌组织中的表达与是否有颈部淋巴结转移、浸润深度以及AJCC分期密切相关(P<0.01),而与年龄、性别、肿瘤的大小以及分化程度无关(P>0.05)。P53在PTC组织中的表达与组织分化程度、是否有淋巴结转移、浸润深度以及AJCC分期密切相关(P<0.01),而与年龄、性别、肿瘤的大小无关(P>0.05)。HSP70和P53蛋白在PTC组织中的表达呈正相关性(r=0.679,P<0.01)。结论:HSP70和p53蛋白在PTC中均呈高表达,并有协同作用,两者可作为预测PTC的生物学行为和预后的参考指标。     

干预DAG-PKC信号转导通路对糖尿病大鼠心肌细胞JNK1和IRS1基因表达的影响

目的干预二酰甘油-蛋白激酶C(DAG-PKC)信号转导通路后观察JNK1、IRS1等在糖尿病大鼠心肌中的表达情况。方法采用HE染色,masson染色、电镜观察大鼠心肌的病理变化,应用免疫组化、Real-Time PCR检测PKCβ2、JNK1及IRS1在大鼠心肌的表达情况。结果糖尿病模型组PKCβ2、JNK1、p-JNK、IRS1表达明显高于对照组,干预DAG-PKC信号转导通路后明显下调其表达水平。结论 DAG-PKC通路可能是通过G蛋白受体和胰岛素受体途径的共同信号点JNK1影响下游的信号传导而导致糖尿病心肌病的发生发展,DAG-PKC-JNK1-IRS1-Akt/PKB-mTOR-p70S6K1等一系列信号位点可能是DAG-PKC信号转导通路引起糖尿病心肌病可能的潜在途径。     

人巨细胞病毒IE1-72蛋白在胶质瘤中的表达及意义

目的:探讨人巨细胞病毒(HCMV)立刻早期基因1-72(IE1-72)蛋白在胶质瘤中的表达水平以及HCMV感染与胶质瘤发生的病因学关系。方法:采用免疫组化方法检测HCMV IE1-72蛋白在125例人脑胶质瘤组织及10例正常人脑组织中的表达,分析其表达水平与胶质瘤临床病理学特征的关系。结果:IE1-72蛋白在胶质瘤组织中的表达水平明显高于正常人脑组织(P=0.000);IE1-72蛋白免疫染色强度随胶质瘤病理级别的升高而明显增强(r=0.310,P=0.000),其在高恶性度胶质瘤中的染色强度明显强于低恶性度胶质瘤(P=0.004);IE1-72蛋白染色强度与胶质瘤患者的年龄存在正相关(r=0.234,P=0.009),而与胶质瘤患者的性别(r=0.038,P=0.675)以及肿瘤部位(r=0.086,P=0.341)无明显相关性。结论:HCMV感染及其蛋白IE1-72表达可能与人脑胶质瘤的发生和发展密切相关,但其确切的致瘤机制尚需进一步研究。     

Anxa2, Stat3 在口腔鳞状细胞癌中的表达及意义

目的:探讨膜联蛋白A2(Anxa2)和细胞信号转导和转录激活因子3(Stat3)在口腔鳞癌组织中的表达及其临床意义。方法:选择口腔鳞癌石蜡标本80例为实验组,20例正常口腔黏膜组织为对照组。应用免疫组织化学法检测Anxa2和Stat3蛋白的阳性表达,并进行结果判定,采用x2和Spearman等级相关分析法分析二者表达差异性及其相关性。结果:Anxa2、Stat3在病例组中的阳性表达率,分别为81.3%(65/80)、87.5%(70/80),明显高于对照组中的表达率,25.0%(5/20)、30.0%(6/20),且差异有统计学意义(P0.05),Anxa2和Stat3蛋白表达水平与口腔鳞癌的分期、淋巴结转移及分化程度有密切关系。Spearman等级相关分析Anxa2和Stat3在口腔鳞癌组织中蛋白的表达呈正相关(r=0.302,P0.01)。结论:Anxa2、Stat3在OSCC的发生和转移过程中均具有重要的作用,且二者间也有相互作用的关系。     

基于电镜和免疫组化的线粒体数目标志物评估肝癌的初步研究

目的:研究线粒体数目与肝癌生长的相关性。方法:利用电子显微镜技术定量肝癌组织中线粒体数目,利用免疫组织化学技术评估肝癌组织中的线粒体标志物COX Ⅳ及HSP60表达水平,分析电镜肝癌线粒体定量结果与COX Ⅳ及HSP60表达量之间的相关性,并比较肝癌中线粒体数目、COX Ⅳ及HSP60表达量与肝癌直径之间的关系。结果:与癌旁相比,肝癌组织中线粒体数目(P0.001)、COX Ⅳ及HSP60的表达量显著降低(P=0.0417,P=0.0290)。COX Ⅳ及HSP60表达水平与线粒体数目无显著相关(r~2=0.009,P=0.5468;r~2=0.056,P=0.1396)。线粒体数目与肿瘤直径显著负相关(r~2=0.1086,P=0.0434),COX Ⅳ及HSP60表达量与肿瘤直径无显著相关(r~2=0.0251,P=0.3287;r~2=0.0461,P=0.1830)。结论:线粒体数目是潜在的肝癌生长标志物。但常用的线粒体定量分子HSP60与COX Ⅳ并不能准确定量肝癌线粒体。     

Fascin 蛋白在软骨肉瘤中的表达及其临床意义

目的：分析Fascin 蛋白在软骨肉瘤组织中的表达及其与软骨肉瘤临床病理因素的关系。方法：应用免疫组化EnlivionTM 法 分别检测和比较70 例软骨肉瘤和20 例软骨瘤中Fascin 蛋白的表达情况。结果：(1) Fascin 在软骨肉瘤组和软骨瘤组中的阳性表 达率分别为64.29%和5.00%,两组相比差异有统计学意义(X2= 21.88,P=0.00);(2)Fascin 蛋白的表达与患者的年龄、性别无显著相 关性(X2= 0.37,P=0.54;X2=0.29,P=0.59);Fascin 蛋白在肺转移组中的表达为76.32%,显著高于无肺转移组(50.00%),两组相比差 异有统计学意义(X2=5.24,P=0.02);Fascin 蛋白在间叶性软骨肉瘤、透明细胞软骨肉瘤的中表达分别与普通型软骨肉瘤相比,无统 计学意义(P>0.05),但去分化软骨肉瘤与普通型软骨肉瘤相比,有统计学意义(P<0.05);Fascin 在普通型软骨肉瘤的Ⅰ-Ⅲ级中的阳 性表达率分别为11.11%、61.90%、75.00%,三者之间的差异有统计学意义(P<0.05)。结论：Fascin 参与了软骨肉瘤恶变的过程,其高 表达与软骨肉瘤的分化和转移有关。     

骨肉瘤中P53及P21WAF1表达的生物学意义

应用免疫组化SP法观察34例骨肉瘤中P53及P21WAF1的表达情况,探讨P53及P21WAF1表达与骨肉瘸临床病理学特征之间的关系,分析P53和P21WAF1在骨肉瘤中的作用及其相关性。结果可见,在34例骨肉瘤中,18例表达P53,阳性表达率52.94%;12例表达P21WAF1,阳性表达率35.29%。P53阳性表达与性别、肿瘤分化及是否转移复发有关(P<0.05),P21WAF1与肿瘤分化有关(P<0.01)。P53在中、低分化骨肉瘤中阳性表达率较高,而在高分化骨肉瘤中阳性表达率较低(70%、28.57%);P21WAF1在高分化骨肉瘤中阳性表达率较高,而在中、低分化骨肉瘤阳性表达率较低(85.71%、0%)。P53及P21WAF1表达呈负相关性(r=-0.537,P=0.001)。在骨肉瘤组织中P53表达上调及P21WAF1表达下调与骨肉瘤的恶性进展有关。     

非小细胞肺癌组织中PTEN基因与Ki67的表达及意义

目的:探讨第10染色体同源丢失性磷酸酶-张力蛋白酶基因(PTEN)、Ki67在非小细胞肺癌组织中的表达及其相关性.方法:用免疫组化方法检测67例非小细胞肺癌组织以及41例癌旁正常肺组织中PTEN基因、Ki67蛋白的表达,并分析其与各临床病理指标及细胞增殖之间的相关性.结果:PTEN基因在67.16％(45/67)的非小细胞肺癌中阳性表达率为32.84％(22/67),而在正常肺组织中阳性表达率为82.97％,显著高于非小细胞癌组织.PTEN基因的表达与组织类型、细胞分化程度、淋巴结转移密切相关(分别为X2=5.44,P=0.019;X2=4.740,P=0.029;X2=4.51,P=0.034),在鳞癌和低分化癌中PTEN基因失表达率或表达减少率较高.肺癌中Ki67的过度表达与肺癌的临床分期、淋巴结转移相关(X2=6.90,P=0.009; X2=5.68,P=0.017),PTEN蛋白阳性表达与Ki67负相关(r=-0.239,P＜0.05).细胞增殖指数越高,PTEN基因表达越少,二者呈负相关(r=-0.252,P＜0.05).结论:PTEN蛋白表达的缺失与非小细胞肺癌的恶性侵袭有关.联合检测PTEN与Ki67的表达可有助于判断非小细胞肺癌的预后.     

HSP70和P53蛋白在甲状腺乳头状癌中的表达及其与临床病理特征的关系

目的：探讨热休克蛋白70（HSP70）和P53蛋白在甲状腺乳头状癌组织中的表达及其与临床病理特征的关系。方法：应用免疫组织化学Envision法检测35例甲状腺乳头状癌组织、15例甲状腺良性病变组织及15例正常甲状腺组织中HSP70和P53蛋白的表达,分析HSP70和P53蛋白的表达与甲状腺乳头状癌临床病理学特征的关系。结果：甲状腺乳头状癌组织中HSP70和P53阳性表达率明显高于甲状腺良性病变组织及正常甲状腺组织（P〈0．01）。HSP70在甲状腺乳头状癌组织中的表达与是否有颈部淋巴结转移、浸润深度以及AJCC分期密切相关（P〈0．01）,而与年龄、性别、肿瘤的大小以及分化程度无关（P〉0．05）。P53在PTC组织中的表达与组织分化程度、是否有淋巴结转移、浸润深度以及AJCC分期密切相关（P〈0．01）,而与年龄、性别、肿瘤的大小无关（P〉0．05）。HSP70和P53蛋白在PTC组织中的表达呈正相关性（r=0．679,P〈0．01）。结论：HSP70和p53蛋白在PTC中均呈高表达,并有协同作用,两者可作为预测PTC的生物学行为和预后的参考指标：     

人脑胶质瘤中热休克蛋白70、Caspase-3 的表达及临床意义

目的:探讨热休克蛋白70、caspase-3在人脑胶质瘤中的表达和临床意义。方法:收集2008年7月～2010年12月汕头大学医学院第二附属医院神经外科手术获取的人脑胶质瘤组织标本35例,另选择20例脑外伤手术中切除的正常脑组织标本作为对照组。采用EnVision免疫组化方法检测脑胶质瘤组织和正常脑组织中热休克蛋白70、caspase-3的表达,并分析其与脑胶质瘤组织临床病理特征之间的关系。结果:HSP70在胶质瘤组和对照组中的阳性表达率分别为74.3%和25.0%,胶质瘤组的HSP70表达明显高于对照组(P<0.05)。caspage-3在胶质瘤组和对照组中的阳性表达率分别为34.3%和80.0%,胶质瘤组的caspage-3阳性表达率均明显低于对照组(P<0.01)。HSP70和caspase-3的阳性表达与胶质瘤的病理学分级、术后复发情况密切相关(P<0.05)。相关分析表明,胶质瘤组织中HSP70阳性表达率和caspase-3表达呈负相关(r=-0.568,P<0.05)。结论:胶质瘤组织中HSP70呈高表达,而caspase-3表达下调,两者均在胶质瘤浸润、复发等恶性演进过程中发挥重要作用;HSP70可能通过某些途径抑制caspase-3表达来抑制胶质瘤恶性细胞的凋亡发生。     

Phosphorylated Heat Shock Protein 20 (HSPB6) Regulates Transforming Growth Factor-α-Induced Migration and Invasion of Hepatocellular Carcinoma Cells

Human hepatocellular carcinoma (HCC) is one of the major malignancies in the world. Small heat shock proteins (HSPs) are reported to play an important role in the regulation of a variety of cancer cell functions, and the functions of small HSPs are regulated by post-translational modifications such as phosphorylation. We previously reported that protein levels of a small HSP, HSP20 (HSPB6), decrease in vascular invasion positive HCC compared with those in the negative vascular invasion. Therefore, in the present study, we investigated whether HSP20 is implicated in HCC cell migration and the invasion using human HCC-derived HuH7 cells. The transforming growth factor (TGF)-α-induced migration and invasion were suppressed in the wild-type-HSP20 overexpressed cells in which phosphorylated HSP20 was detected. Phospho-mimic-HSP20 overexpression reduced the migration and invasion compared with unphosphorylated HSP20 overexpression. Dibutyryl cAMP, which enhanced the phosphorylation of wild-type-HSP20, significantly reduced the TGF-α-induced cell migration of wild-type HSP20 overexpressed cells. The TGF-α-induced cell migration was inhibited by SP600125, a c-Jun N-terminal kinases (JNK) inhibitor. In phospho-mimic-HSP20 overexpressed HuH7 cells, TGF-α-stimulated JNK phosphorylation was suppressed compared with the unphosphorylated HSP20 overexpressed cells. Moreover, the level of phospho-HSP20 protein in human HCC tissues was significantly correlated with tumor invasion. Taken together, our findings strongly suggest that phosphorylated HSP20 inhibits TGF-α-induced HCC cell migration and invasion via suppression of the JNK signaling pathway.     

(-)-Epigallocatechin gallate reduces transforming growth factor beta-stimulated HSP27 induction through the suppression of stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts

We previously reported that transforming growth factor-beta (TGF-beta) stimulates heat shock protein 27 (HSP27) induction through p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether (-)-epigallocatechin gallate (EGCG), the major polyphenol found in green tea, affects the TGF-beta-stimulated induction of HSP27 in these cells, and its underlying mechanism. EGCG significantly suppressed the HSP27 induction stimulated by TGF-beta in a dose-dependent manner between 10 and 30 microM without affecting the HSP70 levels. TGF-beta with or without EGCG did not affect the advanced oxidation protein products. The TGF-beta-induced phosphorylation of p38 MAP kinase and ERK1/2 was not affected by EGCG. SP600125, a specific inhibitor of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), markedly reduced the HSP27 expression induced by TGF-beta. EGCG significantly suppressed the TGF-beta-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of Smad2. EGCG attenuated the phosphorylation of both MKK4 and TAK1 induced by TGF-beta. These results strongly suggest that EGCG suppresses the TGF-beta-stimulated induction of HSP27 via the attenuation of the SAPK/JNK pathway in osteoblasts, and that this effect is exerted at a point upstream from TAK1.     

不同热休克蛋白在肾细胞癌组织中的表达及其意义

目的　研究HSPgp96、HSP90、HSP70在肾细胞癌组织中的表达及其相互关系。方法　应用免疫组化S P法检测 5 4例肾细胞癌组织标本中HSPgp96、HSP90、HSP70的表达情况。结果　在肾细胞癌组织中HSPgp96、HSP90、HSP70表达明显较正常肾组织增强 (P <0 0 5 ) ,阳性率分别为 88 9%、 85 2 %、 90 7% ,三者之间无明显差异性 (P >0 0 5 ) ,其表达程度均与肾细胞癌临床分期、病理分级及细胞类型无显著相关性 (P >0 0 5 )。结论　HSPgp96、HSP90、HSP70的高表达在肾细胞癌的发生、发展中起促进作用 ,尤其是在加强肿瘤抗原的呈递、表达上具有重要临床应用价值。     

Suppression by heat shock protein 20 of hepatocellular carcinoma cell proliferation via inhibition of the mitogen‐activated protein kinases and AKT pathways

Heat shock protein (HSP) 20, one of the low‐molecular weight HSPs, is known to have versatile functions, such as vasorelaxation. However, its precise role in cancer proliferation remains to be elucidated. While HSP20 is constitutively expressed in various tissues including the liver, we have previously reported that HSP20 protein levels in human hepatocellular carcinoma (HCC) cells inversely correlate with the progression of HCC. In this study, we investigated the role of HSP20 in HCC proliferation. The activities of extracellular signal‐regulated kinase (ERK), c‐jun N‐terminal kinase (JNK), and AKT were negatively correlated with the HSP20 protein levels in human HCC tissues. Since HSP20 proteins were hardly detected in HCC‐derived cell lines, the effects of HSP20 expression were evaluated using human HCC‐derived HuH7 cells that were stably transfected with wild‐type human HSP20 (HSP20 overexpressing cells). In HSP20 overexpressing cells, cell proliferation was retarded, and the activation of the mitogen‐activated protein kinases (MAPKs) signaling pathways, including the ERK and JNK, and AKT pathways, as well as cyclin D1 accumulation induced by either transforming growth factor‐α (TGFα) or hepatocyte growth factor, were significantly suppressed compared with the empty vector‐transfected cells (control cells). Taken together, our findings strongly suggest that HSP20 suppresses the growth of HCC cells via the MAPKs and AKT signaling pathways, thus suggesting that the HSP20 could be a new therapeutic target for HCC. J. Cell. Biochem. 112: 3430–3439, 2011. © 2011 Wiley Periodicals, Inc.     

Preheating accelerates mitogen-activated protein (MAP) kinase inactivation post-heat shock via a heat shock protein 70-mediated increase in phosphorylated MAP kinase phosphatase-1

Heat shock (HS) activates mitogen-activated protein (MAP) kinases. Although prior exposure to nonlethal HS makes cells refractory to the lethal effect of a subsequent HS, it is unclear whether this also occurs in MAP kinase activation. This study was undertaken to evaluate the effect of a heat pretreatment on MAP kinase activation by a subsequent HS and to elucidate its possible mechanism. Preheating did not make BEAS-2B cells refractory to extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) activation by a second HS but accelerated their inactivation after HS. The rapid inactivation of ERK and JNK was dependent on de novo protein synthesis and associated with the up-regulation of heat shock protein 70 (HSP70). Moreover, the inhibition of phosphatase activity reversed this rapid inactivation. MAP kinase phosphatase-1 (MKP-1) expression was increased by HS, and the presence of its phosphorylated form (p-MKP-1) correlated with the observed rapid ERK and JNK inactivation. Blocking induction of p-MKP-1 with antisense MKP-1 oligonucleotides suppressed the rapid inactivation of ERK and JNK in preheated cells. HSP70 overexpression caused the early phosphorylation of MKP-1. Moreover, MKP-1 phosphorylation and the rapid inactivation of ERK were inhibited by blocking HSP70 induction in preheated cells. In addition, MKP-1 was insolubilized by HS, and HSP70 associated physically with MKP-1, suggesting that a chaperone effect of HSP70 might have caused the early phosphorylation of MKP-1. These results indicate that preheating accelerated MAP kinase inactivation after a second HS and that this is related to a HSP70-mediated increase in p-MKP-1.     

Protein Kinase R Modulates c-Fos and c-Jun Signaling to Promote Proliferation of Hepatocellular Carcinoma with Hepatitis C Virus Infection

Double-stranded RNA-activated protein kinase R (PKR) is known to be upregulated by hepatitis C virus (HCV) and overexpressed in hepatocellular carcinoma (HCC). However, the precise roles of PKR in HCC with HCV infection remain unclear. Two HCV replicating cell lines (JFH-1 and H77s), generated by transfection of Huh7.5.1 cells, were used for experiments reported here. PKR expression was modulated with siRNA and a PKR expression plasmid, and cancer-related genes were assessed by real-time PCR and Western blotting; cell lines were further analyzed using a proliferation assay. Modulation of genes by PKR was also assessed in 34 human HCC specimens. Parallel changes in c-Fos and c-Jun gene expression with PKR were observed. Levels of phosphorylated c-Fos and c-Jun were upregulated by an increase of PKR, and were related to levels of phosphorylated JNK1 and Erk1/2. DNA binding activities of c-Fos and c-Jun also correlated with PKR expression, and cell proliferation was dependent on PKR-modulated c-Fos and c-Jun expression. Coordinate expression of c-Jun and PKR was confirmed in human HCC specimens with HCV infection. PKR upregulated c-Fos and c-Jun activities through activation of Erk1/2 and JNK1, respectively. These modulations resulted in HCC cell proliferation with HCV infection. These findings suggest that PKR-related proliferation pathways could be an attractive therapeutic target.