气道上皮细胞的抗氧化损伤保护及微环境调控

本工作建立了臭氧对原代培养的兔支气管上皮细胞（ＢＥＣ）损伤模型,观察到血管活性肠肽（ＶＩＰ）、表皮生长因子（ＥＧＦ）、热应激等微环境理化因子可减轻细胞损伤,具有细胞保护作用,其保护机制与提高还原谷胱甘肽（ＧＳＨ）含量有关,并依赖于蛋白激酶的磷酸化调节及基因转录。ＢＥＣ细胞在基础情况下有ｂｃｌ－２基因的低水平表达。ＶＩＰ和ＥＧＦ可促进ｂｃｌ－２基因转录,增强ＢＥＣ的抗氧化损伤能力。ＥＧＦ或热应激促进     

重组RA—538及反义c—myc腺病毒对人胃癌,食管癌和bcl—2高表达细胞系的作用

本课题研究ＲＡ５３８、反义c-ymc重组腺病毒对人胃癌（ＳＧＣ７９０１）、食管癌（Ｅ Ｃ１０９、ＥＣ８７１２）、正常人胚肺２ＢＳ（２ＢＳ）及ｂｃｌ－２高表达细胞第的体仙外生物学作用及其分子机制。结果显示Ａｄ－ＲＡ５３８及Ａｄ－ＡＳｃ－ｍｙｃ对ＳＧＣ７９０１细胞体内外均具有明显的生长抑制及凋亡诱导作用,并能抑制其ｃ－ｍｙｃ、ｂｃｌ－２、ｃｙｃｌｉｎＤ１基因的表达及刺激ｂａｘ基因的表达。对ＥＣ１０９、ＥＣ８     

慢性缺氧大鼠肺血管c-myc及bcl-2基因表达研究

应用免疫组织化学和Ｎｏｒｔｈｅｒｎ杂交方法,对慢性缺氧大鼠肺组织（主要是肺血管壁）原癌基因ｃ－ｍｙｃ和抗凋亡基因ｂｃｌ－２表达进行了研究。免疫组织化学观察提示,正常对照组大鼠肺血壁Ｃ－ｍｙｃ和Ｂｃｌ－２蛋白仅为弱阳性或不表达,慢性缺氧１、２周组大鼠肺血管壁这两种抗原表达比对照组显著增强,呈强阳性,Ｎｏｒｔｈｅｒｎ杂交结果表明：慢性缺氧１、２周组大鼠肺组织内ｃ－ｍｙｃ和ｂｃｌ－２的ｍＲＮＡ表达比对照组显著增加,以上结果提示,ｃ－ｍｙｃ及ｂｃｌ－２两种基因调节的细胞增殖与凋亡可能参与了慢性缺氧性肺动脉高压的发病进程。     

核酶对bcl－2mRNA的体外切割

通过微机对ｂｃｌ－２ＲＮＡ二级结构的分析,设计针对ｂｃｌ－２片段５＇ＣＧＣＧＡＣＣＣＧＧＵＣＧＣＣＡＧＧＡＣＣＵＣＧ３＇的“锤头状”（Ｈａｍｍｅｒｈｅａｄ）核酶（Ｒｉｂｏｚｙｍｅ,ＲＤ）基因,平端连接于ｐＧＥＭ－３Ｚｆ（－）ＨｉｎｃⅡ位点,克隆后经测序表明序列正确,ｂｃｌ－２和Ｒｉｂｏｚｙｍｅ基因经体外转录,５０℃作用２ｈ,从１６５６－１６５７（Ｃ－Ｇ）位之间切断ｂｃｌ－２ＲＮＡ片段．     

碱性成纤维细胞生长因子对大鼠肾小球系膜细胞原癌基因c-fos和c-myc表达的影响

在建立大鼠肾小球系膜细胞（ＭＣ）体外培养方法的基础上,通过３Ｈ－ＴｄＲ参入实验,ＲＮＡ印迹分析和斑点杂交观察ｂＦＧＦ对ＭＣＤＮＡ合成及原癌基因ｃ－ｆｏｓ和ｃ－ｍｙｃ表达的影响．结果表明,ｂＦＧＦ作用于ＭＣ１８ｈ,ＭＣ的３Ｈ－ＴｄＲ参入率明显增加（Ｐ＜０．０５）,２４ｈ达到高峰（Ｐ＜０．０１）;ｂＦＧＦ显著诱导原癌基因ｃ－ｆｏｓ和ｃ－ｍｙｃ表达,其表达活性分别于３０ｍｉｎ和１ｈ达到高峰．提示ｂＦＧＦ是ＭＣ的强效丝裂原,其对ＭＣＤＮＡ合成的促进作用与诱导原癌基因ｃ－ｆｏｓ和ｃ－ｍｙｃ表达有关．     

乙型肝炎、肝硬变和肝癌中HBxAg的表达及其在肝癌发生中的作用

对３７９例良、恶性肝组织进行的免疫组织化学研究显示,３３％的慢性迁延性肝炎（６／１８）、７６％的慢性活动性肝炎（２６／３４）、９２％的肝硬变（５７／６２）和９７％的肝细胞性肝癌（ＨＣＣ）（５８／６０）中有ＨＢｘＡｇ表达,阳性率高于ＨＢｓＡｇ或ＨＢｃＡｇ。癌周肝中的ＨＢｘＡｇ阳性率显著高于非癌周肝。与其它２种ＨＢＶ抗原不同,ＨＢｘＡｇ表达在细胞类型上有较明显的选择性,在肝小多角细胞（ＳＰＬＣ）、小细胞性不典型增生（ＳＣＤ）及ＨＣＣ中较强。与ＩＧＦⅡ、ｃ－ｅｒｂＢ－２、ｃ－ｍｙｃ和ＥＧＦ－Ｒ表达进行的对照研究表明ＨＢｘＡｇ与ＩＧＦⅡ和ｃ－ｅｒｂＢ－２这２种ＨＣＣ发生相关基因的表达关系密切。ＰＣＮＡ染色结果显示ＨＢｘＡｇ阳性组织的细胞增殖活性显著高于ＨＢｘＡｇ阴性组织。我们的结果还表明ＨＢｘＡｇ表达与肝细胞不典型增生的发生和进展有关、提出ＨＢＶＸ基因可能通过其表达产物（ＨＢｘＡｇ）首先激活ＩＧＦⅡ、ｃ－ｅｒｂＢ－２基因,继而引起显著的ＳＰＬＣ增生和ＳＣＤ而参与ＨＣＣ发生的．     

细胞松驰素B促微丝解聚对DNA合成的作用

利用微丝（ｍｉｃｒｏｆｉｌａｍｅｎｔ,ＭＦ）解聚药物细胞松驰素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ,ＣＢ）处理Ｇ_０期小鼠Ｃ_３Ｈ_（１０）Ｔ_（１／２）成纤维细胞,对Ｇ_０至Ｓ期ＤＮＡ合成,胸腺嘧啶核苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ,ＴＫ）活性、ＴＫ基因表达、钙调素（ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）水平和一些细胞周期早期基因的表达进行了观察,Ｇ_０期细胞经３ｍｇ／ＬＣＢ处理２ｈ,促ＭＦ解聚增强了血清对Ｓ期细胞ＴＫ活性、ＴＫ基因表达和ＤＮＡ合成的刺激作用,并促进细胞提前进入Ｓ期．血清刺激Ｇ_０期细胞进入晚Ｇ_１期和Ｓ期时,ＣａＭ水平明显升高,而ＣＢ预处理则使ＣａＭ含量进一步增加,特别是ＣＢ处理促使Ｓ期ＣａＭ增加向核内转移．ＣＢ处理明显增强血清对ｃ－ｊｕｎ、ｃ－ｆｏｓ和ｃ－ｍｙｃ基因表达的刺激作用,而ＰＫＣ抑制剂Ｈ_７则抑制ＣＢ处理对这些基因转录的刺激作用,说明ＣＢ使Ｇ_０期细胞ＭＦ解聚刺激ｃ－ｊｕｎ、ｃ－ｆｏｓ和ｃ－ｍｙｃ的转录活性与ＰＫＣ的作用有关．结果表明Ｇ_０至Ｓ期早期ＭＦ的重组可促进细胞进入Ｓ期,增强ＤＮＡ合成．     

细胞凋亡过程中bcl-2基因的甲基化

为探讨凋亡过程中,ｂｃｌ－２基因下调与该基因甲基化状态的关系,用５－氟尿嘧啶（５－Ｆｕ）诱导小鼠成纤维细胞ＮＣ３Ｈ１０,ＴＣ３Ｈ１０及人乳腺癌细胞ＭＣＦ－７的凋亡,分别检测了这三种细胞凋亡过程中ｂｃｌ－２的表达变化,与其调控区及编码区的甲基化状况．我们曾观察到５－Ｆｕ作用２４～４８ｈ出现细胞存活率下降,ＤＮＡ梯状断裂及细胞周期凋亡峰显现等典型凋亡现象．Ｎｏｒｔｈｅｒｎ杂交显示,在５－Ｆｕ作用１２ｈ时ｂｃｌ－２ｍＲＮＡ水平已明显降低．由此,我们用小鼠ｂｃｌ－２（ｍｂｃｌ－２）及人ｂｃｌ－２（ｈｂｃｌ－２）基因调控区ＰＣＲ扩增片段及ｂｃｌ－２编码区（ｃＤＮＡ）片段作为探针,与５－Ｆｕ作用１２ｈ的细胞ＤＮＡ的ＭｓｐⅠ／ＨｐａⅡ酶切产物进行Ｓｏｕｔｈｅｒｎ杂交,以未作用的细胞ＤＮＡ同样酶切杂交为对照．通过杂交带谱的变化,分析ｂｃｌ－２基因的甲基化状况．结果显示：ｍｂｃｌ－２及ｈｂｃｌ－２在５－Ｆｕ作用１２ｈ后调控区甲基化水平增高,但其编码区甲基化状态皆未出现可检出的变化．上述结果提示：ｂｃｌ－２基因调控区甲基化水平升高可能与该基因下调有关     

DDPH对猪肺动脉平滑肌细胞PDGF·BB和bFGF表达的影响：免疫细胞化学研究

ＤＤＰＨ［１－（２．６－二甲基苯乙氧基）－２－（３．４二甲氧基苯乙胺基）丙烷盐酸盐］是南京药科大学合成的降压新化合物,也具有降低肺动脉高压和抑制肺动脉平滑肌细胞增殖作用。本实验用细胞培养、免疫细胞化学、图像分析、３Ｈ－ＴｄＲ、细胞周期测定等方法,进一步探讨ＤＤＰＨ对缺氧性肺动脉平滑肌细胞（ＰＡＳＭＣＳ）增殖的抑制机制。结果：缺氧促进肺动脉内皮细胞（ＰＡＥＣｓ）的ＰＤＧＦ·ＢＢ和ｂＦＧＦ两种生长因子的表达（积分光密度ＯＤ值）增高。缺氧内皮细胞条件培养液（ＨＥＣＣＭ）能促进ＰＡＳＭＣＳ的ＰＤＧＦ·ＢＢ的ＯＤ值增高,ｂＦＧＦ的ＯＤ值无明显改变。加药组（ＨＥＣ－ＣＭ＋ＤＤＰＨ）的ＰＤＧＦ·ＢＢ和ｂＦＧＦ的ＯＤ值均显著降低,尤以ＰＤＧＦ·ＢＢ的ＯＤ值减少最多．提示：ＤＤＰＨ能抑制ＨＥＣＣＭ引起ＰＡＳＭＣＳ的ＰＤＧＦ·ＢＢ和ｂＦＧＦ表达增多和细胞增殖。结果与大鼠实验观察相符。     

人表皮生长因子(EGF)与单核-巨噬细胞集落刺激因子(GM-CSF)融合蛋白在大肠杆菌中的表达

应用基因工程技术,将ＥＧＦ、ＧＭ－ＣＳＦ基因克隆到ｐＧＥＭ－３Ｚｆ（＋）载体的ＥｃｏＲＩ,ＢａｍＨＩ位点上,再将重组融合基因亚克隆到表达载体ｐＢＶ２２０的ＥｃｏＲＩ,ＢａｍＨＩ位点上,在大肠杆菌ＤＨ５α中进行表达,ＳＤＳ－聚丙烯酰胺凝胶电泳和Ｗｅｓｔｅｒｎｂｌｏｔ表明ＥＧＦ－ＧＭ－ＣＳＦ融合蛋白获得表达,并且具有ＥＧＦ、ＧＭ－ＣＳＦ的免疫学活性．这为进一步研究该融合蛋白的功能和肿瘤治疗提供一种新的基因产品．     

肺内调节肽对兔支气管上皮细胞与嗜酸性粒细胞粘附功能的影响及机制探讨

为了探讨肺内调节肽在各类过敏性炎症发生,发展中的作用,我们观察了血管活性肠肽（vasoactive intestinal peptide,VIP)、表皮生长因子（epidermal growth factor,EGF)、内皮素-1（endothelin-1,ET-1)、降钙素基因相关肽（calcitonin gene-related peptide,CGRP)在未受应激与臭氧应激两种条件下对支气管上皮细胞（bronchial epithelial cell,BEC)与嗜酸性粒细胞（eosinophil,EOS)粘附的影响,结果发现,VIP、EGF可使O3应激的BEC与EOS的粘附率下降,下调气道上皮炎症反应：ET-1、CGRP可使未受应激的BEC与EOS的粘附率增加,诱发炎症损伤反应;CGRP还能加重臭氧的应激反应;ET-1、CGRP的效应可被W7、H7阻断,抗细胞间粘附分子-1（intercel-lular adhesion molecule,ICAM-1)抗体能阻断BEC与EOS的粘附,提示介导BEC与EOS粘附的粘附分子可能是ICAM-1。     

牛c-myc基因的克隆及其在皮肤成纤维细胞中的表达

为了构建包含牛c-myc基因编码序列的重组载体,以胎牛原始生殖嵴为材料,用RT-PCR方法克隆出牛c-myc 基因的编码序列,将其亚克隆至pMD19-T载体,再从酶切鉴定和测序正确的质粒上切下目的片段,定向克隆到pIRES2-AcGFP1-Nuc表达载体上,挑选序列正确的重组真核表达质粒转染牛皮肤成纤维细胞,用RT-PCR和Western blotting分别检测c-myc mRNA和蛋白的表达。结果表明,从胎牛原始生殖嵴中正确克隆了c-myc基因的全长编码序列,所构建的重组质粒能够在皮肤成纤维细胞中有效     

Modification of gene expression by dietary antioxidants in radiation-induced apoptosis of mice splenocytes

The modification of radiation-induced apoptosis in splenocytes by a vitamin-containing dietary supplement was studied. For 45 days prior to irradiation at a lethal dose of 6 Gy, mice received a dietary supplement containing vitamins with antioxidant properties and microelements. The expression of TRPM-2 (a marker for programmed cell death), bcl-2 (the product of which has been shown to prevent apoptosis), superoxide dismutase, and catalase genes was studied at different time intervals after irradiation. Radiation-induced alterations in gene expression were different in the control and the antioxidant mixture-fed mice. The antioxidant mixture administration resulted in an inhibition of TRPM-2 expression both before and after irradiation. The bcl-2 mRNA content steadily increased after irradiation in splenocytes from antioxidant mixture-fed mice, while in the control group 2-h after irradiation only trace amount of bcl-2 mRNA was detected. In splenocytes from control mice, the expression of superoxide dismutase and catalase genes significantly decreased within 2-h after irradiation; whereas in mice receiving the antioxidant mixture, inhibition of catalase gene expression was not as prominent. The expression of superoxide dismutase gene was still high 24-h after irradiation. The antioxidant administration decreased the radiation-induced apoptosis and delayed internucleosomal fragmentation of DNA. Our data suggest that radiation-induced alteration of gene expression is, at least in part, determined by reactive oxygen species.     

siRNA抑制c—myc基因的表达对宫颈癌细胞增殖的影响

目的：利用siRNA（small interference RNA）技术研究C-myc基因的对宫颈癌HeLa细胞增殖的影响。方法：依据Promega公司在网上提供的设计软件,设计针对C-myc基因的siRNA,合成DNA模板,体外转录合成siRNA。通过阳离子聚合物jet—SITM—ENDO将合成的siRNA转染入HeLa细胞,以未转染细胞以及错义序列siRNA—scr转染细胞为对照。用细胞计数法检测siRNA对HeLa细胞增殖的影响。流式细胞法检测细胞周期及蛋白表达的变化,RT—PCR法比较转染前后C-myc mRNA表达水平的变化。结果：细胞计数法结果显示,转染24h后c-myc基因siRNA明显抑制MCF-7细胞增殖,转染48h后,抑制效率稳定。c-myc基因siRNA转染后能有效地抑制HeLa细胞的增殖,阻滞细胞周期于G0／G1期,siRNA转染组c-myc mRNA、蛋白的表达量明显低于空白对照组、错义序列组。结论：体外转录合成的siRNA可有效降低HeLa细胞c-myc基因的表达,抑制细胞增殖。     

Estrogen receptor-alpha, bcl-2 and c-myc gene expression in fibroadenomas and adjacent normal breast: association with nodule size, hormonal and reproductive features

Fibroadenomas are the most common benign lump in females. The study of gene alterations and/or deregulation in reproductive years may help explain hormonal physiological processes involved in nodule development and evolution. The objective was to compare ER-alpha, c-myc, and bcl-2 gene expression in breast fibroadenomas and in normal tissue and evaluate menstrual cycle, parity, and oral contraceptive influences. Fifty-seven premenopausal women (14-49 years) undergoing surgical removal of fibroadenomas were selected. Samples from fibroadenomas and circumjacent normal tissue were obtained for RT-PCR paired analysis. Patients were divided in groups according to menstrual cycle, use of contraceptives and parity. Tissue from 32 patients was adequate for RT-PCR. Paired analysis showed higher expression of ER-alpha (P=0.012) and bcl-2 (P=0.001) in fibroadenomas than in normal breast, while c-myc presented a similar expression (P=0.655). ER-alpha was higher in fibroadenomas of patients in follicular phase versus contraceptive users and normal tissue (P=0.003); bcl-2 was higher in fibroadenomas of patients in luteal phase than in the normal samples from all groups (P=0.007). c-myc did not differ according to menstrual cycle, but was higher in fibroadenomas>3 cm versus<3 cm (P=0.015) and in nulliparous women (P=0.04). A positive correlation between c-myc levels and fibroadenoma diameter was demonstrated (r=0.536; P=0.007). Nulliparous mean nodule diameter was superior than parous women (P=0.008). In conclusion, the expression of ER-alpha, bcl-2 and c-myc depends on hormonal and reproductive factors, with a possible contribution to lump formation and evolution.     

P2Y receptors activate neuroprotective mechanisms in astrocytic cells

Mechanical or ischemic trauma to the CNS causes the release of nucleotides and other neurotransmitters into the extracellular space. Nucleotides can activate nucleotide receptors that modulate the expression of genes implicated in cellular adaptive responses. In this investigation, we used human 1321N1 astrocytoma cells expressing a recombinant P2Y2 receptor to assess the role of this receptor in the regulation of anti-apoptotic (bcl-2 and bcl-xl) and pro-apoptotic (bax) gene expression. Acute treatment with the P2Y2 receptor agonist UTP up-regulated bcl-2 and bcl-xl, and down-regulated bax, gene expression. Activation of P2Y2 receptors was also coupled to the phosphorylation of cyclic AMP responsive element binding protein that positively regulates bcl-2 and bcl-xl gene expression. Cyclic AMP responsive element decoy oligonucleotides markedly attenuated the UTP-induced increase in bcl-2 and bcl-xl mRNA levels. Activation of P2Y2 receptors induced the phosphorylation of the pro-apoptotic factor Bad and caused a reduction in bax/bcl-2 mRNA expression ratio. All these signaling pathways are known to be involved in cell survival mechanisms. Using cDNA microarray analysis and RT-PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor 2; neurotrophin 3; glia-derived neurite-promoting factor, as well as extracellular matrix proteins CD44 and fibronectin precursor--genes known to regulate neuroprotection. Consistent with this observation, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Taken together, our results suggest an important novel role for the P2Y2 receptor in survival and neuroprotective mechanisms under pathological conditions.     

肺内调节肽对支气管上皮细胞ICAM-1表达及NF-κB活性的调控

细胞间粘附分子—1(ICAM—1)是介导细胞与细胞之间粘附的重要生物分子;核因子—κB(NF—κB)是体内普遍存在、能迅速对刺激产生反应的重要核转录因子。越来越多的证据显示,ICAM—1表达与NF—κB激活是炎症反应的重要步骤。我们应用免疫组化、RT—PCR、凝胶阻滞电泳(EMSA)等多种实验方法,观察了肺内调节肽对支气管上皮细胞ICAM—1表达及NF—κB活性的影响,以及NF—κB抑制剂MG—132对ICAM—1表达的影响。实验结果发现,VIP、EGF可使臭氧应激BECS的ICAM—1表达降低;ET—1、CGRP可使未受应激BECs的ICAM—1表达增加。NF—κB抑制剂MG—132可阻断O3、ET—1、CGRP引起的ICAM—1表达,提示NF—κB在调控ICAM—1表达中起重要作用。EMSA结果显示,BECs中NF—κB在臭氧应激下反复激活,CGRP与ET—1可促进NF—κB的激活;VIP与EGF可抑制臭氧应激的BECs中NF—κB的激活。以上结果说明,VIP、EGF可通过下调ICAM—1转录及NF—κB激活减轻炎症反应,而ET—1、CGRP可通过上调ICAM—1转录及NF—κB激活、加大炎症反应。ICAM—1与NF—κB的持续表达和反复激活是炎症持续加重发展的重要因素。