E-cd在胃癌、癌旁组织及转移淋巴结中的表达

研究E-cd在胃癌、癌旁组织及转移淋巴结中的表达。用免疫组化方法检测E-cd的表达,用图像分析仪进行定量分析。结果显示胃癌及转移淋巴结中E－cd的表达明显低于癌旁组织（P＜0.001）。分化不良胃癌中E－cd的表达明显低于分化良好胃癌中的表达（P＜0.05）。结果表明E-cd可作为判断胃癌预后的指标。     

降钙素基因相关肽对人支气管上皮细胞E-钙粘素表达的影响

目的:探讨降钙素基因相关肽(CGRP)对臭氧(O3)应激后人支气管上皮细胞(HBECs)中E-钙粘素(E-cd)表达的影响及机制。方法:采用RT-PCR检测E-cd mRNA的表达,免疫细胞化学染色法检测E-cd蛋白的表达。结果:CGRP呈剂量依赖性增加正常以及O3应激后HBECs胞膜上E-cd的表达,而对胞浆内E-cd的表达无明显影响;CGRP对HBECs胞膜上E-cd表达的上调作用可分别被H-89(PKA抑制剂)、H-7(PKC抑制剂)及W-7(CaM抑制剂)部分逆转。结论:CGRP可剂量依赖性增加正常和O3应激后HBECs胞膜E-cd的表达,而对胞浆内E-cd的表达无影响。     

吸烟小鼠呼吸道上皮细胞上皮钙粘附素表达的研究

通过免疫荧光法检测不同吸烟时间小鼠呼吸道上皮细胞上皮钙粘附素（Ｅｃａｄｈｅｒｉｎ,Ｅｃｄ）表达的动态变化,并采用显微荧光光度术进行定量分析。结果表明,首次吸烟１ｈ后呼吸道上皮细胞Ｅｃｄ表达与正常对照组比较明显下调（Ｐ＜００１）,４６Ｗ后降到最低水平。以后逐渐上调,吸烟８Ｗ后Ｅｃｄ表达恢复正常,气管上皮细胞表达高于正常（Ｐ＜００１）。提示,Ｅｃｄ表达的动态变化在呼吸道上皮细胞损伤与修复过程中起重要作用     

香烟烟雾提取物抑制肺泡上皮细胞的增殖并诱导其凋亡

香烟烟雾提取物（cigarette smoke extract,CSE）中含有丰富的氧化剂和自由基,由它所引起的氧化应激可导致肺泡壁的损伤进而发展为肺气肿.近年来,围绕CSE损伤肺泡壁作用机制的研究较为活跃,但其结果却一直存在着分歧.本实验的目的是观察CSE对肺泡Ⅱ型上皮细胞的损伤作用并探讨与其相关的分子机制.MTT比色法的结果显示,CSE以时间和剂量依赖性的方式降低细胞的增殖活力,流式细胞术的分析结果表明细胞增殖周期被阻滞在G1/S期.Hoechst 33258染色以及透射电镜观察从形态上确认CSE诱导细胞凋亡的发生,DNA梯的出现和Annexin V-FITC/碘化丙啶双染色的结果从分子水平得到进一步的证实.同时,运用流式细胞术检测到CSE诱导的凋亡伴随着Fas受体的高表达和caspase-3的显著活化.另外,使用H2DCFDA染色,经激光共聚焦显微镜术测得细胞内氧自由基在细胞受到CSE刺激以后大量快速积累.结果表明CSE能够抑制肺泡Ⅱ型上皮细胞来源的A549细胞的生长和增殖,并诱导细胞凋亡,由Fas受体所介导的死亡受体途径参与此凋亡过程,而CSE所引起的氧化应激则可能是阻止肺泡上皮细胞生长增殖并诱导其凋亡的始动因素.     

口腔疣状癌和口腔鳞癌组织中E-cadherin表达的对比研究

目的:研究E-钙粘素(E-cadherin,E-cd)在口腔疣状癌和口腔鳞癌中的表达,探讨E-cd与口腔疣状癌,口腔鳞癌分化程度的关系。方法:采用免疫组织化学和免疫电镜方法检测18例口腔疣状癌、20例口腔鳞癌组织中的E-cd表达。结果:E-cd在正常口腔黏膜、口腔疣状癌、高分化鳞癌和低分化鳞癌中主要位于胞膜及胞浆上。电镜观察显示E-cd在胞膜主要定位于桥粒,胞浆主要定位于粗面内质网和线粒体上。定量检测显示:E-cd在正常口腔黏膜、口腔疣状癌、高分化鳞癌和低分化鳞癌中的阳性表达率分别100%(8/8)、61.1%(11/18)、50.0%(5/10)和30.0%(3/10)。口腔疣状癌E-cd蛋白的平均染色强度与高分化鳞癌间差异无显著性,但明显低于正常黏膜组(P<0.05)而高于低分化鳞癌(P<0.05)。结论:E-cd的表达水平随口腔肿瘤分化程度的降低而降低,且超微结构的表达部位与肿瘤的分化程度存在相关性。     

无赖氨酸激酶4在小鼠气管上皮细胞液体转运中的作用

该研究探讨了无赖氨酸激酶4(with no lysine kinase 4, WNK4)对于小鼠气管上皮细胞液体转运中的调节作用。在原代培养的小鼠气管上皮细胞中,应用siRNA特异性沉默WNK4基因,半定量PCR和Western blot实验验证沉默效率;随后应用尤斯灌流装置和Western blot实验记录该激酶的低表达对小鼠气管上皮细胞的短路电流及钠离子通道α-亚基蛋白表达水平的影响。半定量PCR和Western blot结果显示,该研究选用的siRNA序列可以沉默WNK4的表达。尤斯灌流和Western blot结果显示,沉默该激酶后,小鼠气管上皮细胞的阿米洛利敏感性电流和钠离子通道α-亚基蛋白表达明显增加。该研究表明,降低WNK4基因表达能增加小鼠气管上皮细胞的上皮钠离子通道α-亚基蛋白表达,促进钠离子转运,此过程可能参与相关水肿性肺疾患的修复。     

去势大鼠阴茎海绵体胱硫醚γ裂解酶与硫化氢的表达

目的观察去势大鼠阴茎海绵体胱硫醚γ裂解酶（Cystathionine-γ-lyase,CSE）/硫化氢（Hydrogen sulphide,H2S）的变化,进一步探讨勃起功能障碍的发病机制。方法雄性SD大鼠72只分4组：对照组、假手术组,去势组和去势炔丙基甘氨酸（PAG,CSE阻断剂）组,检测基础条件下和阿扑吗啡（Apomorphine,APO）刺激后的海绵体内压（Intracavernous pressure,ICP）及勃起率;激光共聚焦显微镜检测CSE在大鼠勃起不同时期阴茎海绵体组织中的表达,敏感硫电极测定H2S在勃起不同时期的含量。结果与假手术组比较,去势组和去势PAG组ICP与勃起率下降（P〈0.01）;且去势PAG组较去势组ICP明显下降（P〈0.01）;阿朴吗啡刺激后,与假手术组比较,勃起前去势组和去势PAG组CSE蛋白表达降低（P〈0.01）,勃起中去势各组较假手术组CSE蛋白表达降低（P〈0.01）,且去势PAG组较去势组CSE蛋白表达明显降低（P〈0.01）;勃起后各组间CSE蛋白表达变化无差异。勃起前和勃起中去势各组较假手术组H2S含量下降（P〈0.05）,且勃起中去势PAG组较去势组H2S含量明显下降（P〈0.01）：勃起后去势组和去势PAG组较假手术组H2S含量下降（P〈0.01）。结论去势大鼠勃起功能障碍与CSE和H2S表达下降有关。     

支气管哮喘小鼠模型气道血管变化及其影响因子分析

目的明确支气管哮喘时血管网络及血管内皮细胞变化,以及血管内皮生长因子（VEGF）亚型及其受体在支气管哮喘小鼠模型气管及肺组织中的变化及作用。方法在建立小鼠支气管哮喘模型的基础上,应用免疫荧光染色和HE染色观察气道旁血管密度变化并计数不同血管单位长度上内皮细胞数量,应用逆转录多聚酶链反应（RT-PCR）方法检测气管和肺组织血管内皮生长因子（VEGF）亚型及其受体mRNA表达情况。结果1.随着疾病时间的延长,小鼠气道壁血管密度增加,血管内皮细胞数量逐渐增加,特别在慢性期小血管尤为明显;2.应用RT-PCR技术及琼脂糖凝胶电泳,小鼠气管及肺组织VEGF120,VEGF164,VEGF188和VEGF205各个亚型mRNA均增高。其中VEGF164在气管组织,VEGF188在周围肺组织中mRNA表达在慢性哮喘期明显增高,少见的亚型VEGF144只在周围肺组织中检测到,而在支气管组织中无表达;VEGFR1 mRNA水平没有明显变化,而VEGFR2在正常气管组织中未检测到,而在支气管哮喘气管组织明显表达,并且在支气管哮喘慢性期高于急性期。结论随着疾病的进展,小鼠支气管哮喘时气道存在血管密度及血管内皮细胞数量增加,VEGF在此过程中起着重要的作用,各亚型的作用存在差异。另外,VEGFR2在支气管哮喘中对于介导和增强VEGF信号和VEGF活性起着重要的作用,并且可能是导致VEGF诱导小鼠气道血管再生与重塑的重要介质。     

腺苷A2a受体在博莱霉素诱导的小鼠肺纤维化中的作用

目的：观察腺苷A_2a受体（A_2aR）在小鼠肺纤维化形成中的调控作用。方法：30只雄性SPF级野生型BALB/C小鼠和20只A_2aR基因敲除BALB/C小鼠,随机分为以下5组：野生型小鼠对照组（A组）、野生型小鼠纤维化组（B组）、A_2aR基因敲除小鼠对照组（C组）、A_2aR基因敲除小鼠纤维化组（D组）、野生型小鼠纤维化+A_2aR激动剂（CGS21680）组（E组）,每组各10只。纤维化组小鼠气管内注入博莱霉素（bleomycin,BLM）溶液50μl （5 mg/kg体重）,对照组在气管内注入等体积生理盐水,注射后立即将小鼠直立旋转3~5 min,使其均匀分布于两肺。A、B、C、D各组每天腹腔注射生理盐水0.5 ml,E组每天腹腔注射A_2aR激动剂（CGS21680）0.5 ml （0.25 mg/kg体重）,连续28 d。第29天取血检测血清转化生长因子β1（TGF-β1）含量;检测肺组织中羟脯氨酸（hydroxyproline,Hyp）、TGF-β1和A_2aR蛋白质含量,TGF-β1 mRNA、A_2aR mRNA的表达,并观察肺组织光镜和超微结构。结果：①光镜和超微结构提示,B组肺泡壁增厚破坏,肺泡腔狭窄或部分陷闭,纤维增生,炎细胞浸润,I型、Ⅱ型肺泡上皮细胞明显空泡化,D组较B组明显,E组表现较B组减轻。Masson染色提示B组、D组小鼠肺组织纤维增生明显,E组明显减少,提示基因缺失加重了小鼠肺纤维化的形成。②与A组比较,B组血清TGF-β1含量,肺组织Hyp含量,TGF-β1、A_2aR蛋白质含量和TGF-β1 mRNA、A_2aR mRNA的表达均明显增高（P<0.01,P<0.05）。与C组比较,D组血清TGF-β1含量、肺组织Hyp、TGF-β1含量和TGF-β1 mRNA表达明显增高（P<0.01,P<0.05）,提示肺纤维化小鼠肺组织Hyp含量增高,TGF-β1表达上调。③与B组相比,D组血清TGF-β1、肺组织Hyp、TGF-β1含量和TGF-β1 mRNA表达明显增高（P<0.01）。与B组比较,E组A_2aR蛋白质含量和A_2aR mRNA的表达均增高（P<0.01）,而血清TGF-β1、肺组织Hyp和TGF-β1蛋白含量及TGF-β1 mRNA表达较B组明显下降（P<0.01,P<0.05）,提示A_2aR基因敲除小鼠肺组织Hyp含量增高、TGF-β1表达上调;A_2aR激动剂可使A_2aR表达上调、TGF-β1表达下降。结论：A_2aR基因敲除小鼠的肺纤维化明显增加。A_2aR在肺纤维化形成中反应性增高,可通过降低TGF-β1的表达而抑制肺纤维化形成。     

Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

Background

Although individuals exposed to cigarette smoke are more susceptible to respiratory infection, the effects of cigarette smoke on lung defense are incompletely understood. Because airway epithelial cell responses to type II interferon (IFN) are critical in regulation of defense against many respiratory viral infections, we hypothesized that cigarette smoke has inhibitory effects on IFN-γ-dependent antiviral mechanisms in epithelial cells in the airway.

Methods

Primary human tracheobronchial epithelial cells were first treated with cigarette smoke extract (CSE) followed by exposure to both CSE and IFN-γ. Epithelial cell cytotoxicity and IFN-γ-induced signaling, gene expression, and antiviral effects against respiratory syncytial virus (RSV) were tested without and with CSE exposure.

Results

CSE inhibited IFN-γ-dependent gene expression in airway epithelial cells, and these effects were not due to cell loss or cytotoxicity. CSE markedly inhibited IFN-γ-induced Stat1 phosphorylation, indicating that CSE altered type II interferon signal transduction and providing a mechanism for CSE effects. A period of CSE exposure combined with an interval of epithelial cell exposure to both CSE and IFN-γ was required to inhibit IFN-γ-induced cell signaling. CSE also decreased the inhibitory effect of IFN-γ on RSV mRNA and protein expression, confirming effects on viral infection. CSE effects on IFN-γ-induced Stat1 activation, antiviral protein expression, and inhibition of RSV infection were decreased by glutathione augmentation of epithelial cells using N-acetylcysteine or glutathione monoethyl ester, providing one strategy to alter cigarette smoke effects.

Conclusions

The results indicate that CSE inhibits the antiviral effects of IFN-γ, thereby presenting one explanation for increased susceptibility to respiratory viral infection in individuals exposed to cigarette smoke.     

Caspase-12 Silencing Attenuates Inhibitory Effects of Cigarette Smoke Extract on NOD1 Signaling and hBDs Expression in Human Oral Mucosal Epithelial Cells

Cigarette smoke exposure is associated with increased risk of various diseases. Epithelial cells-mediated innate immune responses to infectious pathogens are compromised by cigarette smoke. Although many studies have established that cigarette smoke exposure affects the expression of Toll-liked receptor (TLR), it remains unknown whether the nucleotide-binding oligomerization domain-containing protein 1 (NOD1) expression is affected by cigarette smoke exposure. In the study, we investigated effects of cigarette smoke extract (CSE) on NOD1 signaling in an immortalized human oral mucosal epithelial (Leuk-1) cell line. We first found that CSE inhibited NOD1 expression in a dose-dependent manner. Moreover, CSE modulated the expression of other crucial molecules in NOD1 signaling and human β defensin (hBD) 1, 2 and 3. We found that RNA interference-induced Caspase-12 silencing increased NOD1 and phospho-NF-κB (p-NF-κB) expression and down-regulated RIP2 expression. The inhibitory effects of CSE on NOD1 signaling can be attenuated partially through Caspase-12 silencing. Intriguingly, Caspase-12 silencing abrogated inhibitory effects of CSE on hBD1, 3 expression and augmented induced effect of CSE on hBD2 expression. Caspase-12 could play a vital role in the inhibitory effects of cigarette smoke on NOD1 signaling and hBDs expression in oral mucosal epithelial cells.     

Cigarette Smoke Extract Induces a Phenotypic Shift in Epithelial Cells; Involvement of HIF1α in Mesenchymal Transition

In COPD, matrix remodeling contributes to airflow limitation. Recent evidence suggests that next to fibroblasts, the process of epithelial-mesenchymal transition can contribute to matrix remodeling. CSE has been shown to induce EMT in lung epithelial cells, but the signaling mechanisms involved are largely unknown and subject of this study. EMT was assessed in A549 and BEAS2B cells stimulated with CSE by qPCR, Western blotting and immunofluorescence for epithelial and mesenchymal markers, as were collagen production, cell adhesion and barrier integrity as functional endpoints. Involvement of TGF-β and HIF1α signaling pathways were investigated. In addition, mouse models were used to examine the effects of CS on hypoxia signaling and of hypoxia per se on mesenchymal expression. CSE induced EMT characteristics in A549 and BEAS2B cells, evidenced by decreased expression of epithelial markers and a concomitant increase in mesenchymal marker expression after CSE exposure. Furthermore cells that underwent EMT showed increased production of collagen, decreased adhesion and disrupted barrier integrity. The induction of EMT was found to be independent of TGF-β signaling. On the contrary, CS was able to induce hypoxic signaling in A549 and BEAS2B cells as well as in mice lung tissue. Importantly, HIF1α knock-down prevented induction of mesenchymal markers, increased collagen production and decreased adhesion after CSE exposure, data that are in line with the observed induction of mesenchymal marker expression by hypoxia in vitro and in vivo. Together these data provide evidence that both bronchial and alveolar epithelial cells undergo a functional phenotypic shift in response to CSE exposure which can contribute to increased collagen deposition in COPD lungs. Moreover, HIF1α signaling appears to play an important role in this process.     

CCN1 Secretion Induced by Cigarette Smoking Extracts Augments IL-8 Release from Bronchial Epithelial Cells

Inflammation involves in many cigarette smoke (CS) related diseases including the chronic obstructive pulmonary disease (COPD). Lung epithelial cell released IL-8 plays a crucial role in CS induced lung inflammation. CS and cigarette smoke extracts (CSE) both induce IL-8 secretion and subsequently, IL-8 recruits inflammatory cells into the lung parenchyma. However, the molecular and cellular mechanisms by which CSE triggers IL-8 release remain not completely understood. In this study, we identified a novel extracellular matrix (ECM) molecule, CCN1, which mediated CSE induced IL-8 secretion by lung epithelial cells. We first found that CS and CSE up-regulated CCN1 expression and secretion in lung epithelial cells in vivo and in vitro. CSE up-regulated CCN1 via induction of reactive oxygen spices (ROS) and endoplasmic reticulum (ER) stress. p38 MAPK and JNK activation were also found to mediate the signal pathways in CSE induced CCN1. CCN1 was secreted into ECM via Golgi and membrane channel receptor aquaporin4. After CSE exposure, elevated ECM CCN1 functioned via an autocrine or paracrine manner. Importantly, CCN1 activated Wnt pathway receptor LRP6, subsequently stimulated Wnt pathway component Dvl2 and triggered beta-catenin translocation from cell membrane to cytosol and nucleus. Treatment of Wnt pathway inhibitor suppressed CCN1 induced IL-8 secretion from lung epithelial cells. Taken together, CSE increased CCN1 expression and secretion in lung epithelial cells via induction of ROS and ER stress. Increased ECM CCN1 resulted in augmented IL-8 release through the activation of Wnt pathway.     

STAT3 activation inhibits human bronchial epithelial cell apoptosis in response to cigarette smoke exposure

We have previously reported that cigarette smoke can induce DNA damage in human lung cells without leading to apoptosis or necrosis. In this study, we report that STAT3 is required for the survival of human bronchial epithelial cells (HBECs) following cigarette smoke-induced DNA damage. Cigarette smoke extract (CSE) exposure increases STAT3 phosphorylation (Tyr 705) and DNA binding activity in HBECs. CSE also stimulates IL-6 release and mRNA expression. Anti-IL-6 neutralizing antibody partially blocks STAT3 activation and renders the cells sensitive to CSE-induced DNA damage. Suppression of STAT3 by siRNA results in severe DNA damage and cell death in response to CSE exposure. These findings suggest that STAT3 mediates HBEC survival in response to CSE-induced DNA damage, at least in part, through the IL-6/STAT3 signaling pathway.     

Cigarette smoke extract affects functional activity of MRP1 in bronchial epithelial cells

Cigarette smoke is the principal risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) superfamily of transporters, which transport physiologic and toxic substrates across cell membranes. MRP1 is highly expressed in lung epithelium. This study aims to analyze the effect of cigarette smoke extract (CSE) on MRP1 activity. In the human bronchial epithelial cell line 16HBE14o-, MRP1 function was studied flow cytometrically by cellular retention of carboxyfluorescein (CF) after CSE incubation and MRP1 downregulation by RNA interference (siRNA). Cell survival was measured by the MTT assay. Immunocytochemically, it was shown that 16HBE14o(-) expressed MRP1 and breast cancer resistance protein. Coincubation of CSE IC50 (1.53% +/- 0.22%) with MK571 further decreased cell survival 31% (p, = 0.018). CSE increased cellular CF retention dose dependently from 1.7-fold at 5% CSE to 10.3-fold at 40% CSE (both p < 0.05). siRNA reduced MRP1 RNA expression with 49% and increased CF accumulation 67% versus control transfected cells. CSE exposure further increased CF retention 24% (p = 0.031). A linear positive relation between MRP1 function and CSE-modulating effects (r = 0.99, p =0.089) was shown in untransfected, control transfected, and MRP1 downregulated 16HBE14o- cells analogous to blocking effects with MRP1 inhibitor MK571 (r = 0.99, p = 0.034). In conclusion, cigarette smoke extract affects MRP1 activity probably competitively in bronchial epithelial cells. Inhibition of MRP1 in turn results in higher CSE toxicity. We propose that MRP1 may be a protective protein for COPD development.     

Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear.

Methodology and Principal Findings

Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7). Cigarette smoke extract (CSE) is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC) inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1) and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSE-induced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1 ^−/− mice, which displayed basal airspace enlargement, resisted cigarette-smoke induced autophagy, apoptosis, and emphysema.

Conclusions

We demonstrate a critical role for Egr-1 in promoting autophagy and apoptosis in response to cigarette smoke exposure in vitro and in vivo. The induction of autophagy at early stages of COPD progression suggests novel therapeutic targets for the treatment of cigarette smoke induced lung injury.     

大鼠体内气管损伤修复过程及气管干细胞的定位研究

目的观察大鼠体内气管损伤修复过程,进行气管干细胞的定位.方法应用氟尿嘧啶(5-FU)诱发在体气管上皮损伤,动态观察修复过程;对损伤后气管上皮细胞行Hoechst33342荧光染色,并用RT-PCR法检测ABC转运蛋白ABCG2/bcrp1基因.结果1.5-FU作用30min后大鼠气管上皮细胞绝大部分脱落,可见少量间隔分布的类似裸核的细胞呈钉状位于基底膜上,免疫组化检测增殖细胞核抗原阴性,证明为G0期细胞.其中部分细胞Hoechst33342染色阴性,为侧群(side population,SP)细胞.2.将5-FU 去除3-6h后,上皮细胞形态变为扁平,9-12h 后细胞变为立方,细胞数目逐渐增多,24h上皮细胞数更多,连接成片,可见纤毛,48h 接近恢复假复层纤毛柱状上皮.3.RT-PCR检测ABCG2/Bcrp1阳性反应产物长度为272bp.结论5-FU打击后,残余的G0期气管上皮细胞中含有干细胞.     

核因子相关因子2调节大鼠气道上皮细胞γ-谷氨酰半胱氨酸合成酶表达

红系衍生的核因子相关因子2(Nrf2)是调节抗氧化基因表达的关键转录因子.γ-谷氨酰半胱氨酸合成酶(γ-GCS)是肺内主要的抗氧化基因,但Nrf2如何调节γ-GCS表达目前仍不清楚.该文采用香烟烟雾提取物(CSE)诱导大鼠气道上皮细胞为研究对象,探索Nrf2调节γ-GCS基因表达机制.细胞免疫荧光显示,Nrf2蛋白质在CSE处理1、3和6h组,主要在胞核中表达.细胞免疫化学与Western印迹显示,γ-GCS蛋白质在CSE1、3、6h组表达明显增强,其中Western印迹结果高于对照组(P0.05);Nrf2胞核蛋白质表达增强.p-aPKCι/ζ蛋白质在CSE1、3h组表达增强,与对照组相比差异显著(P0.05).RT-PCR结果表明,γ-GCSmRNA在CSE1、3、6h组表达明显高于对照组(P0.05).γ-GCS活性在CSE1、3、6h组增高并高于对照组(P0.05).GSH含量在CSE3、6h组明显高于对照组(P0.05).预先加入aPKCι/ζ抑制剂RO813220,Nrf2胞浆蛋白质表达增强,GSH含量、p-aPKCι/ζ蛋白质、γ-GCS蛋白质与其mRNA和活性均明显低于CSE3h组(P0.05).相关性分析显示Nrf2与γ-GCS、γ-GCS活性、p-aPKCι/ζ呈正相关,p-aPKCι/ζ与Nrf2、γ-GCS、γ-GCS活性呈正相关(P0.05).以上结果表明,CSE可能通过aPKCι/ζ-Nrf2信号通路调节γ-GCS的表达水平和活性.