吸烟大鼠淋巴细胞蛋白质组的双向电泳分析

目的应用双向凝胶电泳(Two-dimensional gel electrophoresis,2-DE)方法分析烟染毒大鼠外周血淋巴细胞蛋白质组表达的改变。方法雄性Wistar大鼠随机分正常对照组和烟染毒组,烟染毒组分小、中、大剂量,分离各组大鼠的外周血淋巴细胞,提取总蛋白,2-DE分离总蛋白,电泳凝胶银染显色,用ImageMaster 2D Platinum软件对获得的蛋白图谱加以分析,寻找差异蛋白点。结果与正常对照组大鼠图谱比较,烟染毒组图谱中与剂量有相关性变化的差异表达蛋白点有10个,其中6个随烟染毒剂量增加表达下降,4个随烟染毒剂量增加表达增强。结论2-DE可以有效分离大鼠外周血淋巴细胞蛋白质,大鼠烟染毒后淋巴细胞蛋白质的表达发生改变。     

肿瘤的基因治疗前期研究:反转录病毒介导的肿瘤坏死因子α基因抗C6胶质瘤效应

目的探讨反转录病毒介导的肿瘤坏死因子α(TNF-α)基因对C6胶质瘤的抗瘤效应,从而为肿瘤的基因治疗提供基础研究数据.方法将重组TNF-α反转录病毒载体PLJ+TNF转染大鼠C6胶质瘤细胞,ELISA法检测该细胞中目的基因的表达,MTT法检测体外增殖能力;将实验大鼠随机分为实验组和对照组,每组10只,实验组大鼠右股内侧接种转基因2×106 C6细胞,对照组同法同部位接种未转基因C6细胞,常规饲养5周,每周测量肿瘤大小,绘制肿瘤生长曲线,比较两组皮下肿瘤大小并进行统计学分析.结果C6胶质瘤细胞与C6细胞的体外增殖能力比较,差异无显著性意义(P＞0.05);转基因前后TNF-α分泌情况1×106个C6细胞体外培养24 h每毫升上清液分泌TNF-α(2.42±0.76)u;1 ×106个C6胶质瘤细胞体外培养24 h每毫升上清液分泌TNF-α(17.53±2.31)u,两者比较,差异有显著性意义(P＜0.01);动态TNF-α检测显示转基因C6细胞能稳定地表达高水平的TNF-α;大鼠皮下接种肿瘤细胞5周后,实验组3只未见肿瘤生长,其余肿瘤直径为(1.5±0.3)cm,对照组肿瘤直径为(2.4±0.2)cm,两组比较,差异有显著性意义(P＜0.01).结论TNF-α基因转染大鼠C6胶质瘤细胞后能持续分泌高水平的TNF-α,对大鼠C6胶质瘤皮下肿瘤具有明显的抗瘤效应.     

肿瘤的基因治疗前期研究：反转录病毒介导的肿瘤坏死因子α基因抗C6胶质瘤效应☆

目的：探讨反转录病毒介导的肿瘤坏死因子α(TNF-α)基因对C6胶质瘤的抗瘤效应，从而为肿瘤的基因治疗提供基础研究数据。方法：将重组TNF-α反转录病毒载体PLJ+TNF转染大鼠C6胶质瘤细胞，ELISA法检测该细胞中目的基因的表达，MTT法检测体外增殖能力；将实验大鼠随机分为实验组和对照组，每组10只，实验组大鼠右股内侧接种转基因2&;#215;10^6C6细胞，对照组同法同部位接种未转基因C6细胞，常规饲养5周，每周测量肿瘤大小，绘制肿瘤生长曲线，比较两组皮下肿瘤大小并进行统计学分析。结果：C6胶质瘤细胞与C6细胞的体外增殖能力比较，差异无显著性意义(P&;gt;0．05)；转基因前后TNF-α分泌情况：1&;#215;10^6个C6细胞体外培养24h每毫升上清液分泌TNF-α(2．42&;#177;0．76)U；1&;#215;10^6个C6胶质瘤细胞体外培养24h每毫升上清液分泌TNF-α(1753&;#177;2．31)U，两者比较，差异有显著性意义(P&;lt;0．01)；动态TNF-α检测显示：转基因C6细胞能稳定地表达高水平的TNF-α；大鼠皮下接种肿瘤细胞5周后，实验组3只未见肿瘤生长，其余肿瘤直径为(15&;#177;0．3)cm，对照组肿瘤直径为(2．4&;#177;0．2)cm，两组比较，差异有显著性意义(P&;lt;0．01)。结论：TNF-α基因转染大鼠C6胶质瘤细胞后能持续分泌高水平的TNF-α，对大鼠C6胶质瘤皮下肿瘤具有明显的抗瘤效应。     

CT灌注成像观测早期基因治疗大鼠C6胶质瘤疗效

目的 探讨常规MR检查结合CT灌注成像早期观察胶质瘤基因治疗效果的作用.方法 雄性Wistar大鼠50只于右侧尾状核接种C6胶质瘤细胞后随机分为两组,治疗组大鼠于相同位置注入携带融合基因的重组单纯疱疹病毒.接种后第2、3周行MR常规及CT灌注检查及病理检查.结果 第2周对照组与治疗组大鼠C6胶质瘤肿瘤体积差异无统计学意义(P>0.05);而治疗组大鼠的rCBF及rCBV小于对照组(P<0.05).第3周时治疗组大鼠肿瘤体积、rCBF及rCBV均小于对照组(P<0.05).结论 CT灌注成像可早期发现抗血管生成基因治疗对大鼠C6脑胶质瘤局部微循环的影响.     

硫化氢延迟预处理对大鼠心肌蛋白质组学的研究

目的 采用蛋白质组学技术,观察硫化氢延迟预处理对大鼠心肌蛋白质表达的影响.方法 16只SD大鼠按随机数字表法分为生理盐水对照组和硫化氢预处理组,每组8只.尾静脉注射生理盐水和硫化氢预处理后24h建立心肌缺血/再灌注模型(缺血30 min、再灌注120 min).用双向凝胶电泳(2-DE)分离两组大鼠心肌组织蛋白质,2-DE图谱分析后找出表达差异的蛋白质,并用基质辅助激光解析-电离飞行时间质谱(MALDI-TOF-MS)鉴定差异蛋白质.结果 对照组的蛋白质点数平均为929±14,硫化氢组的蛋白质点数平均为906±10,经凝胶图像分析找到表达差异蛋白质15个;用MALDI-TOF-MS对差异蛋白进行鉴定,共鉴定出11个蛋白质,包括多(聚)脱氧核糖核苷酸合酶、胱硫醚-γ-裂解酶、转录起始因子、NADH脱氢酶、鸟嘌呤核苷酸释放因子、果糖二磷酸醛缩酶A、糖原合成酶激酶-3、电子转移黄素蛋白、谷胱甘肽S转移酶、钙活化核苷酸酶、S-腺苷蛋氨酸合成酶.结论 硫化氢延迟预处理可使大鼠心肌组织蛋白表达发生改变,其心肌保护作用可能与改善能量代谢、抗氧化反应、细胞保护和保护呼吸链等有关.     

早期诊断唐氏综合征的母体血清蛋白新标志物筛选与初探验证

目的 探讨经筛选与验证的唐氏综合征(DS)相关特异性母体血清蛋白新标志物的临床应用价值.方法 选取确诊为DS胎儿的孕中期(14 ～ 20周)母体血清7份(DS组)和同期正常胎儿母体血清7份(正常对照组)行二维凝胶电泳(2-DE),建立DS胎儿母体血清蛋白表达差异图谱,提取差异表达蛋白质点进行质谱分析.免疫印迹法(WB)验证部分差异蛋白的表达差异.采用近似t检验比较分析2组间相应蛋白条带的吸光度(A)值差异.结果 经2-DE和差异蛋白图谱分析,在DS组母体血清中共发现表达差异1.5倍以上的蛋白质点29个,其中表达上调的19个,表达下调的10个.表达差异2倍以上的8个蛋白质点经质谱分析,从美国国立生物技术信息中心( NCBI)蛋白质序列数据库中搜索为dGTP酶(dGTPase)和β2-糖蛋白Ⅰ(β2-GPI)等12个蛋白质分别与之匹配的可能性较大(概率得分＞66分).WB证实GTPase和β2 -GPI的A值在DS组分别为21 567.0±3009.4和22 097.0 ± 3958.9,在正常对照组分别为3957.7±250.9和1799.7±105.5,dGTP酶和β2-GPI在DS胎儿母体血清中比正常对照组显著高表达(t ' dGTPasc=- 17.66,t’β2 CPI=- 14.83,P均＜0.0001).结论 2 -DE和质谱技术是初步筛选DS相关特异性母体血清中新蛋白标志物的有效方法,经WB验证的dGTPase和β2 -GPI可能是筛选DS的新标志物.     

海洛因对C6细胞嘌呤核苷酸分解代谢基因表达的影响

目的研究海洛因对C6胶质瘤细胞嘌呤核苷酸分解代谢关键酶基因表达的影响。方法海洛因作用于C6胶质瘤细胞12h、24h、48h、72h后,提取细胞总RNA,采用逆转录聚合酶链反应（RT-PCR）检测嘌呤核苷酸分解代谢的关键酶腺苷脱氨酶（ADA）和黄嘌呤氧化酶（XO）基因表达的变化,以β-actin为内标准。结果在转录物水平,浓度为20μg/ml的海洛因作用于C6细胞24h和48h时与对照组相比ADA、XO基因表达明显增强（P〈0.05）。结论在转录物水平海洛因能一定程度增强C6细胞嘌呤核苷酸分解代谢关键酶基因的表达,从而促进嘌呤核苷酸分解代谢。     

苯乙酸对胶质瘤细胞HOXA1-A4基因mRNA表达的影响

目的观察苯乙酸（PA）诱导分化胶质瘤细胞C6过程中，同源盒基因HoxA1，A2，A3和HoxA4基因mRNA水平表达的变化。方法应用逆转录PCR（RT-PCR）及图像分析法，分组检测原代培养的大鼠星形胶质细胞及应用PA前后胶质瘤细胞C6中HoxA，A2，A3和HoxA4基因mRNA水平表达。结果HoxA1和A4基因在正常大鼠脑组织中弱表达，在胶质瘤C6细胞中存在明显表达；HoxA2基因在正常大鼠脑组织中未见表达，在胶质瘤C6细胞中存在明显表达；HoxA3基因在正常大鼠脑组织和应用PA前后的胶质瘤C6细胞中，均存在明显表达。应用PA处理后，胶质瘤C6细胞中HoxA1、A2基因未见表达，与未用药的C6细胞比较，图象分析显示差异有显著性（P〈0．01）；应用PA处理后，HoxA3、A4基因明显表达，与未用药的C6细胞比较，图象分析显示差异没有显著性（P〉0．05）。结论苯乙酸抑制胶质瘤细胞增殖的作用机理可能与降低胶质瘤细胞HoxA1和HoxA2基因mRNA水平表达有关。     

胶质瘤荷瘤大鼠γ刀照射后C-myc蛋白表达变化及意义

目的　观察胶质瘤Wistar荷瘤大鼠γ刀照射后 ,C myc蛋白表达的变化 ,探讨γ刀的作用机理。方法　建立颅内种植胶质瘤C6细胞的Wistar荷瘤大鼠模型。 3周后 ,实验组给予γ刀治疗。计数荷瘤大鼠生存时间。免疫组织化学SP染色及计算机图像分析检测大鼠C myc基因蛋白水平表达。结果　实验组及对照组荷瘤大鼠生存时间分别为 :36 5± 3 1d ,2 8 0± 2 4d ;实验组生存时间明显延长 (P <0 0 1)。实验组及对照组荷瘤大鼠C myc基因蛋白阳性表达平均百分率分别为 :4 0 98%± 15 4 6 % ,77 6 2 %± 12 11% ;二者差异显著 (P <0 0 1)。结论　γ刀诱导胶质瘤凋亡的机制可能与抑制肿瘤细胞C myc基因蛋白水平表达有关。     

Endostatin-Angiostatin融合基因治疗大鼠C6胶质瘤的3.0T磁共振动态观察

目的：探讨3．OT磁共振在重组单纯疱疹病毒介导的Endostatin—Angiostatin（Endo-Angio）融合基因对大鼠C6胶质瘤疗效评价中的作用。方法：30只雄性Wistar大鼠采用立体定向方法在颅内接种C6细胞，将荷瘤大鼠随机分为两组，治疗组与对照组；两组大鼠分别于接种后1、2、3周进行MR检查；第2、3周检查结束后每组留取2只鼠脑标本进行病理学检查；治疗组大鼠于第1周检查后于肿瘤接种位置原位注入EndoAngio融合基因进行治疗。结果：第1周检查共25只大鼠成瘤（成功率83％），第2周两组肿瘤体积均增大，二者无统计学差异（P〉0．01），病理检查显示治疗组大鼠微血管腔减少，问质水肿及细胞水肿较对照组明显严重；第3周时治疗组大鼠体积减小，小于对照组（P〈O．01）。结论：重组单纯疱疹病毒介导的Endo—Angio融合基因对大鼠C6胶质瘤的生长有抑制作用，3．0T磁共振可以较好地动态观察大鼠C6胶质瘤的生长及治疗变化。     

胶质瘤来源exosome的鉴定及其蛋白质组成研究

目的初步分析胶质瘤细胞分泌的exosome蛋白组成,探讨胶质瘤来源exosome的潜在免疫调节功能,从而为进一步利用exosome对胶质瘤进行免疫治疗提供理论依据。方法采用差速离心法从U251胶质瘤细胞培养上清液和Ⅲ级星形胶质瘤囊液中分别提纯exosome,用透射电镜鉴定;利用二维电泳分离、分析exosome内蛋白质,并用质谱技术鉴定了部分蛋白质。结果胶质瘤细胞可以产生exosome,其平均直径约100 nm。二维电泳图显示U251细胞分泌的exosome含有270个蛋白点,与数据库相符的有66个;而来自Ⅲ级星形胶质瘤囊液的exosome含有242个蛋白点,与数据库相符的有60个;两者有130个蛋白点在等电点和表观分子量方面相同,其中包含HSP70、RNA结合蛋白、核酸外切酶、MHCI及MHCII类分子等。部分蛋白质点质谱鉴定结果为hCG、低密度脂蛋白、T细胞受体等。结论胶质瘤细胞可分泌exosome,其一般特性与已报道的exosome一致,其蛋白组成与其他细胞来源的exosome具有共性,体内和体外培养的胶质瘤细胞分泌的exosome的蛋白质组成具有同源性与差异性。胶质瘤细胞源的exosome具有一定的免疫调节功能,可以为胶质瘤免疫治疗提供理论基础。     

On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells

BACKGROUND: That there is a correlation between cancer and procoagulant states is well-known. C6 glioma cell line was originally induced in random-bred Wistar-Furth rats and is morphologically similar to glioblastoma multiforme, the most common aggressive glioma resistant to therapeutic interventions. OBJECTIVES: In this study we analyzed the molecular mechanisms responsible for the highly procoagulant properties of C6 glioma cells. METHODS: The presence of tissue factor (TF) and phosphatidylserine (PS) in C6 cells was investigated by flow-cytometric and functional analyses. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. RESULTS: TF was identified by flow-cytometric and functional [factor (F) Xa formation in the presence of cells and FVIIa] assays. Alternatively, conversion of FX into FXa was also observed in the presence of C6 cells, FIXa and FVIIIa. This effect was both cell- and FVIIIa-dependent, being consistent with formation of the intrinsic tenase complex. C6 cells were also able to activate prothrombin in the presence of FXa and FVa, thus supporting formation of the prothrombinase complex. This ability was similar to positive controls performed with PS-containing vesicles. Accordingly, exposure of PS on C6 cells was demonstrated by flow cytometry employing specific anti-PS antibodies. In addition, annexin V, which blocks PS binding sites, inhibited FX and prothrombin conversion by their respective C6-assembled activating complexes. CONCLUSION: C6 glioma cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant TF exposure and from the presence of the anionic lipid PS at the outer leaflet of cell membrane. Therefore, this animal cell line may be used to explore new aspects concerning the role of blood coagulation proteins in tumor biology, especially those affecting the central nervous system.     

Increased bax expression is associated with cell death induced by ganciclovir in a herpes thymidine kinase gene-expressing glioma cell line

The herpes simplex virus thymidine kinase gene (HSV-tk) was stably transfected into rat C6 glioma cells (C6tk) in order to characterize the mechanisms underlying cell toxicity induced in vitro by the guanosine analog ganciclovir (GCV). The results demonstrate the efficiency of the HSV-tk/GCV system in ablating most of the tumoral cells within 7 to 8 days of treatment with 20 mivroM GCV; however, a few cells still survive. C6tk cells arrest in the S phase of the cell cycle after 2 days of drug treatment before undergoing cell death. Microscopic analysis reveals dying cells with ultrastructural characteristics consistent with apoptosis; we cannot rule out, however, that necrotic cell death may also be occurring. The cytotoxicity induced by GCV is not associated with changes in the expression of p53 protein, suggesting that cell cycle arrest and cell death may occur through a p53-independent pathway. C6tk cells constitutively express Bcl-xL and Bax proteins; when exposed to GCV, Bcl-xL levels do not change but Bax accumulation is rapidly induced. These findings suggest that the balance between Bcl-xL and Bax proteins may be of importance in determining the sensitivity of tumoral cells to GCV.     

快速埋线对偏头痛肝阳上亢证大鼠下丘脑差异蛋白质表达的影响

目的:探讨快速埋线缓解肝阳上亢型偏头痛大鼠与相关蛋白的机理。方法:30只大鼠随机抽取20只制成肝阳上亢证模型后又复制为实验性偏头痛模型,其中10只给予快速埋线治疗(埋线组);10只给予蒸馏水灌服(上亢组);另10只为正常组。用双向凝胶电泳检测3组大鼠下丘脑的总蛋白质的表达。结果:与正常组匹配,上亢组与埋线组大鼠下丘脑图象匹配率分别为92%、96%,且重复性较好,其蛋白质主要分布在等电点PI4-8,分子量Mr20-75 kD。与正常组比较,上亢组蛋白质点表达上升2倍及以上12个,下降2倍及以上14个;埋线组以上表达上升的点有13个下降2倍以上,其中8个点下降后接近正常组,而表达下降的点均上升,有9个点上升达2倍以上,其中6个点上升后接近于正常组。结论:与正常组差异有显著的蛋白质可能是偏头痛肝阳上亢证证候相关蛋白;快速埋线可缓解头痛和改善临床症状可能与上述蛋白质的改变有关。     

体外血管内皮生长因子单克隆抗体对C6胶质瘤细胞增殖、侵袭和迁移力的影响

目的:在体外观察血管内皮生长因子(VEGF)单克隆抗体对C6胶质瘤细胞增殖、侵袭和迁移力的影响。方法:采用氯化钴(Co Cl2)模拟缺氧环境,予以0、0.1、1、10μg/m L的VEGF抗体,对常氧或缺氧条件下对C6胶质瘤细胞进行处理。通过MTT实验检测细胞增殖,Transwell实验检测细胞的侵袭和迁移能力,Western blotting检测HIF-1α蛋白、FAK/Pyk2总蛋白及磷酸化FAK-Tyr397/Pyk2-Tyr402表达水平变化。结果:200μmol/L Co Cl2明显抑制C6细胞增殖(P<0.05),10μg/m L VEGF抗体可明显抑制C6细胞增殖(P<0.05)。与常氧相比,缺氧可使C6细胞的迁移、侵袭能力增强(P<0.05)。常氧和缺氧下,VEGF抗体的干预均增强C6细胞的迁移、侵袭能力(P<0.05),且相对于常氧,缺氧可进一步增强VEGF抗体促C6细胞迁移、侵袭的作用(P<0.05)。Co Cl2干预可增加C6细胞中HIF-1α的含量;在常氧和缺氧情况下,只有1μg/m L VEGF抗体可降低FAK的总蛋白量(P<0.05);在缺氧情况下,VEGF抗体可明显增高Pyk2-Tyr402位点的磷酸化水平。结论:在缺氧情况下,VEGF抗体的干预可使Pyk2-Tyr402位点的磷酸化水平增高,引起C6细胞的侵袭迁移能力进一步增强。     

联合应用氯喹增加人胶质瘤细胞 U343细胞对 Bcl-2抑制剂 ABT737的敏感性

目的 探讨 Bcl-2抑制剂 ABT737联合氯喹的抗肿瘤作用。方法 选用人胶质瘤细胞 U343细胞系,应用MTT 方法检测 ABT737和（或）氯喹对 U343细胞活性的影响,western blot 检测 ABT737和（或）氯喹对 U343细胞凋亡通路相关蛋白的作用。结果 ABT737显著降低细胞生存率,增加 U343细胞凋亡相关蛋白 Cleaved PARP、Cleaved Caspase-3及细胞色素 C（cytochrome c）表达。当氯喹与 ABT737联用时,二者联用与单用 ABT737组相比,细胞活性显著降低,显著下调凋亡通路相关蛋白 Cleaved PARP、Cleaved Caspase-3、细胞色素 C 表达。结论 联用氯喹可提高人胶质瘤细胞 U343细胞对 Bcl-2抑制剂 ABT737的敏感性。     

Tumor gene therapy made easy: allogeneic major histocompatibility complex in the C6 rat glioma model

The C6 glioma in the immune-competent rat is a frequently used model in brain tumor gene therapy research. It displays the histologic hallmarks of the human glioblastoma and has been employed to demonstrate new mechanisms of anti-tumor immunity and therapeutic strategies. We noted that C6 tumors regressed spontaneously in three of five animals and that protective anti-tumor immunity ensued without therapeutic intervention. A review of the literature revealed that different rat strains are used as "syngeneic" host for the C6 cell glioma, namely, BDIX, BDX, Sprague-Dawley, and Wistar. Allelotyping of the RT1.A (rat MHC I homolog) by a serologic technique and of the RT1.B (rat MHC II homolog) by a newly developed molecular technique showed that C6 cells express the haplotype RT1u and are allogeneic in the preceding rat strains. Expression of the gene encoding the transactivator CIITA in C6 gliomas using an EBV-based transduction system led to induction of MHC I and II and thereby mimicked therapeutic responses that could not operate in syngeneic models. These data suggest that the C6 glioma model in the immune-competent rat should no longer be used to study gene therapy strategies, that the available data obtained in this model need to be critically reinterpreted, and that findings obtained in the C6 glioma model may not be sufficient to support a clinical trial in glioblastoma patients.     

平瘤康方剂对C6胶质瘤细胞增殖的抑制作用

目的探讨中药方剂平瘤康含药血清对体外培养的C6胶质瘤细胞增殖及细胞周期的影响。方法2.5%、5%、10%和20%平瘤康含药血清处理体外培养的C6胶质瘤细胞24h、48h和72h后,应用MTT比色法检测C6胶质瘤细胞增殖;PI染色后,应用流式细胞仪观测细胞周期时相。结果10%和20%的含药血清能抑制C6胶质瘤细胞的增殖;10%和20%的含药血清处理C6细胞48h、72h后,S期细胞百分率下降,呈剂量依赖性。结论平瘤康含药血清可能通过阻滞细胞周期而抑制胶质瘤细胞增殖。     

Proteome of H-411E (liver) cells exposed to insulin and tumor necrosis factor-alpha: analysis of proteins involved in insulin resistance

Insulin resistance may be modeled in H-411E liver cells in tissue culture with the use of the cytokine tumor necrosis factor-alpha (TNF-alpha) and insulin. This tissue-culture model nicely mimics IR in human type 2 diabetes mellitus. After incubation of liver cells in tissue culture with INS alone, TNF-alpha alone, and TNF-alpha plus insulin, as well as a control sample, liver-cell extracts were separated on 2D polyacrylamide-gel electrophoresis on the basis of isoelectric point and molecular weight. We analyzed the gel images with the use of PD Quest software (Bio-Rad Laboratories, Hercules, Calif) to identify differentially expressed protein spots (ie, up or down with insulin vs down or up with TNF-alpha plus insulin). In separate experiments, phosphorus-32 incorporation/autoradiography and phosphoprotein staining were used to characterize treatment-induced phosphorylations. Affected protein spots were identified with the use of peptide fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry. The first series of experiments identified 6 differentially expressed proteins: eukaryotic translation initiation factor-3, subunit 2, regulator of G-protein signaling-5, superoxide dismutase, protein disulfide isomerase A6, proteasome subunit-alpha type 3, and regucalcin. In addition, we observed changes in the phosphorylation of protein disulfide isomerase A6. A second series of experiments identified 7 additional proteins with significantly altered differential expression: cell-division protein kinase-4, kinogen heavy chain, carbonic anhydrase-7, E 3 ubiquitin protein ligase, URE-B1; Rab GDP dissociation inhibitor-beta, Rab GDP dissociation inhibitor-beta2, and MAWDBP. It can be seen that differentially expressed proteins, affected by treatment with insulin or with TNF-alpha plus insulin, include regulators of translation, protein degradation, cellular Ca ++ , G-proteins, and free-radical production. Although one cannot detail the mechanism or mechanisms of TNF-alpha induced IR from this data alone, it is easy to relate all of these proteins to a role in insulin signal transduction and, hence, insulin resistance.